Cancer treatment kits comprising therapeutic conjugates that bind to aminophospholipids

ABSTRACT

Disclosed is the surprising discovery that aminophospholipids, such as phosphatidylserine and phosphatidylethanolamine, are specific, accessible and stable markers of the luminal surface of tumor blood vessels. The present invention thus provides aminophospholipid-targeted diagnostic and therapeutic constructs for use in tumor intervention. Antibody-therapeutic agent conjugates and constructs that bind to aminophospholipids are particularly provided, as are methods of specifically delivering therapeutic agents, including toxins and coagulants, to the stably-expressed aminophospholipids of tumor blood vessels, thereby inducing thrombosis, necrosis and tumor regression.

The present application claims priority to first provisional applicationSer. No. 60/092,589, filed Jul. 13, 1998, and second provisionalapplication Ser. No. 60/110,600, filed Dec. 2, 1998, the entire text andfigures of which applications are incorporated herein by referencewithout disclaimer.

The U.S. Government owns rights in the present invention pursuant togrant numbers 1RO1CA74951-01 and 5RO1CA54168-05 from the NationalInstitutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of blood vesselsand tumor biology. More particularly, it embodies the surprisingfindings that aminophospholipids, such as phosphatidylserine andphosphatidylethanolamine, are accessible, stable and specific markers oftumor vasculature. The invention thus provides therapeutic constructsand conjugates that bind to aminophospholipids for use in deliveringtoxins and coagulants to tumor blood vessels and for inducing thrombosisand tumor regression.

2. Description of the Related Art

Tumor cell resistance to chemotherapeutic agents represents asignificant problem in clinical oncology. In fact, this is one of themain reasons why many of the most prevalent forms of human cancer stillresist effective chemotherapeutic intervention, despite certain advancesin the field of chemotherapy.

A significant problem to address in tumor treatment regimens is thedesire for a “total cell kill”. This means that the more effectivetreatment regimens come closer to a total cell kill of all so-called“clonogenic” malignant cells, i.e., cells that have the ability to growuncontrolled and replace any tumor mass that might be removed by thetherapy. Due to the goal of developing treatments that approach a totalcell kill, certain types of tumors have been more amenable to therapythan others. For example, the soft tissue tumors, e.g., lymphomas, andtumors of the blood and blood-forming organs, e.g., leukemias, havegenerally been more responsive to chemotherapeutic therapy than havesolid tumors, such as carcinomas.

One reason for the susceptibility of soft and blood-based tumors tochemotherapy is the greater accessibility of lymphoma and leukemic cellsto chemotherapeutic intervention. Simply put, it is much more difficultfor most chemotherapeutic agents to reach all of the cells of a solidtumor mass than it is the soft tumors and blood-based tumors, andtherefore much more difficult to achieve a total cell kill. Increasingthe dose of chemotherapeutic agents most often results in toxic sideeffects, which generally limits the effectiveness of conventionalanti-tumor agents.

Another tumor treatment strategy is the use of an “immunotoxin”, inwhich an anti-tumor cell antibody is used to deliver a toxin to thetumor cells. However, in common with the chemotherapeutic approachesdescribed above, immunotoxin therapy also suffers from significantdrawbacks. For example, antigen-negative or antigen-deficient cells cansurvive and repopulate the tumor or lead to further metastases. Also, inthe treatment of solid tumors, the tumor mass is generally impermeableto molecules of the size of antibodies and immunotoxins. Both thephysical diffusion distances and the interstitial pressure within thetumor are significant limitations to this type of therapy.

A more recent strategy has been to target the vasculature of solidtumors. Targeting the blood vessels of the tumors, rather than the tumorcells themselves, has certain advantages in that it is not likely tolead to the development of resistant tumor cells, and that the targetedcells are readily accessible. Moreover, destruction of the blood vesselsleads to an amplification of the anti-tumor effect, as many tumor cellsrely on a single vessel for their oxygen and nutrients (Denekamp, 1990).Exemplary vascular targeting strategies are described in U.S. Pat. Nos.5,855,866 and 5,965,132 (U.S. application Ser. No. 08/350,212, Issue Feepaid), which particularly describe the targeted delivery ofanti-cellular agents and toxins to protein markers of tumor vasculature.

Another effective version of the vascular targeting approach is totarget a coagulation factor to a protein marker expressed or adsorbedwithin the tumor vasculature (Huang et al, 1997; U.S. Pat. Nos.5,877,289, 6,004,555 and 9,093,399) The delivery of coagulants, ratherthan toxins, to tumor vasculature has the further advantages of reducedimmunogenicity and even lower risk of toxic side effects. As disclosedin U.S. Pat. No. 5,877,289, a preferred coagulation factor for use insuch tumor-specific thrombogens, or “coaguligands”, is a truncatedversion of the human coagulation-inducing protein, Tissue Factor (TF).TF is the major initiator of blood coagulation (Ruf et al., 1991;Edgington et al., 1991; Ruf and Edgington, 1994). Treatment oftumor-bearing mice with such coaguligands results in significant tumornecrosis and even complete tumor regression in many animals (Huang etal, 1997; U.S. Pat. Nos. 5,877,289, 6,004,555 and 6,093,399)

Although the specific delivery of therapeutic agents, such asanti-cellular agents, toxins and coagulation factors, to protein markersof tumor vessels represents a significant advance in tumor treatmentprotocols, there is still room for additional vascular targetingtherapies. The identification of additional stable targets to allowspecific tumor vessel destruction in vivo would naturally be of benefitin expanding the number of targeting options. More particularly, thedevelopment of targeting agents for delivering therapeutics even closerto the tumor vascular endothelial cell membrane would represent animportant advance.

SUMMARY OF THE INVENTION

The present invention addresses the needs of the prior art by providingnew compositions and methods for tumor vasculature imaging anddestruction. The invention is based, in part, on the finding thataminophospholipid membrane components, such as phosphatidylserine andphosphatidylethanolamine, are accessible, stable markers of tumorvasculature. The invention thus provides binding ligands and antibodiesagainst aminophospholipids that are operatively attached to therapeuticagents, and methods of using constructs in the specific delivery ofdiagnostics and therapeutics to the actual surface of tumor vascularendothelial cell membranes.

Important aspects of the invention are that therapeutic agents can bedelivered in intimate contact with the tumor vascular endothelial cellmembrane, allowing either rapid entry into the target cell or rapidassociation with effector cells, components of the coagulation cascade,and such like. Certain surprising features of the invention include thediscovery that translocation of aminophospholipids, such asphosphatidylserine (PS), to the surface of tumor vascular endothelialcells occurs, at least in a significant part, independently of celldamage and apoptopic or other cell-death mechanisms. Thus, PS surfaceexpression in this environment is not a consequence of cell death, nordoes it trigger immediate cell destruction.

The discovery of sufficiently stable PS expression on morphologicallyintact tumor-associated vascular endothelial cells is important to thetargeting nature of the present invention. Should PS translocation tothe outer surface of tumor vascular endothelium occur only in dyingcells, or should it inevitably trigger cell death, then PS expressionwould be expected to be transient and PS would not likely be a goodcandidate target for therapeutic intervention. Surprisingly, the presentinvention shows that significant stable PS expression occurs in viableendothelial cells in a tumor environment, thus providing ample targetingopportunities.

The present invention therefore basically provides methods fordelivering selected diagnostic and therapeutic agents to tumor orintratumoral vasculature, comprising administering to an animal having avascularized tumor a biologically effective amount of a binding ligandthat comprises a selected diagnostic or therapeutic agent operativelyattached to a targeting agent that binds to an aminophospholipid,preferably one that binds to phosphatidylserine orphosphatidylethanolamine, on the luminal surface of blood vessels orintratumoral blood vessels of the vascularized tumor.

The methods of the invention provide for killing, or specificallykilling, tumor or intratumoral vascular endothelial cells, and compriseadministering to an animal or patient having a vascularized tumor abiologically effective amount of at least a first pharmaceuticalcomposition comprising a binding ligand that comprises a selectedtherapeutic agent operatively attached to a targeting agent that bindsto an aminophospholipid, preferably one that binds to phosphatidylserineor phosphatidylethanolamine, on the luminal surface of tumor orintratumoral vascular endothelial cells.

The “binding ligands” of the present invention are thus“aminophospholipid binding ligands”, “therapeutic aminophospholipidbinding ligand constructs”, “aminophospholipid-targeted therapeuticagents”, “aminophospholipid-targeted therapeutics”,“aminophospholipid-targeted therapeutic agent constructs”, or“therapeutic agent-aminophospholipid targeting agent constructs”. Forsimplicity, these agents are referred to herein as “binding ligands” or“therapeutic agent-targeting agent constructs”, with the understandingthat such terms are used as a succinct way of referring to a conjugateor other operative association of a selected therapeutic agent and atargeting agent, antibody, binding protein or active fragment thereof,that binds to an aminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, expressed on the luminal surface of tumor orintratumoral vascular endothelial cells.

“Biologically effective amounts” are amounts of the therapeuticagent-targeting agent construct effective to specifically kill at leasta portion, and preferably a significant portion, of the tumor orintratumoral vascular endothelial cells, as opposed to endothelial cellsin normal vessels, upon binding to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, expressed on the luminalsurface of the tumor or intratumoral vascular endothelial cells. Assuch, it is an “endothelial cell killing amount” or a “tumor vascularendothelial cell killing amount” of a therapeutic agent-targeting agentconstruct.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,except in instances wherein an upper limit is thereafter specificallystated. Therefore a “therapeutic agent-targeting agent construct” means“at least a first therapeutic agent-targeting agent construct”. Theoperable limits and parameters of combinations, as with the amounts ofany single agent, will be known to those of ordinary skill in the art inlight of the present disclosure.

The “a” and “an” terms are also used to mean “at least one”, “at least afirst”, “one or more” or “a plurality” of steps in the recited methods,except where specifically stated. This is particularly relevant to theadministration steps in the treatment methods. Thus, not only maydifferent doses be employed with the present invention, but differentnumbers of doses, e.g., injections, may be used, up to and includingmultiple injections.

An “aminophospholipid”, as used herein, means a phospholipid thatincludes within its structure at least a first primary amino group.Preferably, the term “aminophospholipid” is used to refer to a primaryamino group-containing phospholipid that occurs naturally in mammaliancell membranes. However, this is not a limitation on the meaning of theterm “aminophospholipid”, as this term also extends to non-naturallyoccurring or synthetic aminophospholipids that nonetheless have uses inthe invention, e.g., as an immunogen in the generation ofanti-aminophospholipid antibodies (“cross-reactive antibodies”) that dobind to aminophospholipids of mammalian plasma membranes. Theaminophospholipids of U.S. Pat. No. 5,767,298, incorporated herein byreference, are appropriate examples.

The prominent aminophospholipids found in mammalian biological systemsare the negatively-charged phosphatidylserine (“PS”) and the neutral orzwitterionic phosphatidylethanolamine (“PE”), which are thereforepreferred aminophospholipids for targeting by the present invention.However, the invention is by no means limited to the targeting ofphosphatidylserines and phosphatidylethanolamines, and any otheraminophospholipid target may be employed (White et al., 1978;incorporated herein by reference) so long as it is expressed, accessibleor complexed on the luminal surface of tumor vascular endothelial cells.

All aminophospholipid-, phosphatidylserine- andphosphatidylethanolamine-based components are encompassed as targets ofthe invention irrespective of the type of fatty acid chains involved,including those with short, intermediate or long chain fatty acids, andthose with saturated, unsaturated and polyunsaturated fatty acids.Preferred compositions for raising antibodies for use in the presentinvention may be aminophospholipids with fatty acids of C18, with C18:1being more preferred (Levy et al., 1990; incorporated herein byreference). To the extent that they are accessible on tumor vascularendothelial cells, aminophospholipid degradation products having onlyone fatty acid (lyso derivatives), rather than two, may also be targeted(Qamar et al., 1990; incorporated herein by reference).

Another group of potential aminophospholipid targets include, forexample, phosphatidal derivatives (plasmalogens), such asphosphatidalserine and phosphatidalethanolamine (having an ether linkagegiving an alkenyl group, rather than an ester linkage giving an acylgroup). Indeed, the targets for therapeutic intervention by the presentinvention include any substantially lipid-based component that comprisesa nitrogenous base and that is present, expressed, translocated,presented or otherwise complexed in a targetable form on the luminalsurface of tumor vascular endothelial cells, not excludingphosphatidylcholine (“PC”). Lipids not containing glycerol may also formappropriate targets, such as the sphingolipids based upon sphingosineand derivatives.

The biological basis for including a range of lipids in the group oftargetable components lies, in part, with the observed biologicalphenomena of lipids and proteins combining in membranous environments toform unique lipid-protein complexes. Such lipid-protein complexes extendto antigenic and immunogenic forms of lipids such as phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine with, e.g., proteinssuch as β₂-glycoprotein I, prothrombin, kininogens and prekallikrein.Therefore, as proteins and polypeptides can have one or more freeprimary amino groups, it is contemplated that a range of effective“aminophospholipid targets” may be formed in vivo from lipid componentsthat are not aminophospholipids in the strictest sense. Nonetheless, allsuch targetable complexes that comprise lipids and primary amino groupsconstitute an “aminophospholipid” within the scope of the presentinvention.

The inventive methods also act to arrest blood flow, or specificallyarrest blood flow, in tumor vasculature. This is achieved byadministering to an animal or patient having a vascularized tumor atleast one dose of at least a first pharmaceutical composition comprisinga coagulation-inducing amount, or a vessel-occluding amount, of at leasta first cytotoxic or coagulative agent operatively attached to atargeting agent that binds to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, translocated to theluminal surface of tumor vasculature.

The “coagulation-inducing amount” or “vessel-occluding amount” is anamount of the therapeutic agent-targeting agent construct effective tospecifically promote coagulation in, and hence occlude, at least aportion, and preferably a significant portion, of tumor or intratumoralblood vessels, as opposed to normal blood vessels, upon binding to anaminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, translocated to the luminal surface of tumoror intratumoral blood vessels. The “vessel-occluding amount” istherefore a functionally effective amount, and is not a physical mass oftherapeutic agent-targeting agent construct sufficient to span thebreadth of a vessel.

Methods for destroying, or specifically destroying, tumor vasculatureare provided that comprise administering to an animal or patient havinga vascularized tumor one or more doses of at least a firstpharmaceutical composition comprising a tumor-destructive amount of atleast a first occluding or destructive agent operatively attached to atargeting agent that binds to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, presented on the luminalsurface of tumor or intratumoral vasculature. The “tumor-destructiveamount” is an amount of the therapeutic agent-targeting agent constructeffective to specifically destroy or occlude at least a portion, andpreferably a significant portion, of tumor or intratumoral bloodvessels, as opposed to normal blood vessels, upon binding to anaminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, presented on the luminal surface of thevascular endothelial cells of the tumor or intratumoral blood vessels.

The invention further encompasses methods for treating cancer and solidtumors, comprising administering to an animal or patient having avascularized tumor a tumor necrosis-inducing amount or amounts of atleast a first pharmaceutical composition comprising at least a firsttherapeutic or necrotic agent operatively attached to a targeting agentthat binds to an aminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, on the luminal surface of blood vessels orintratumoral blood vessels of the vascularized tumor. The “tumornecrosis-inducing amount” is an amount of the therapeuticagent-targeting agent construct effective to specifically inducehemorrhagic necrosis in at least a portion, and preferably a significantportion, of the tumor upon binding to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, complexed at the luminalsurface of the vascular endothelial cells of the tumor or intratumoralblood vessels, while exerting little adverse side effects on normal,healthy tissues.

The methods of the invention may thus be summarized as methods fortreating an animal or patient having a vascularized tumor, comprisingadministering to the animal or patient a therapeutically effectiveamount of at least a first pharmaceutical composition comprising atleast a first therapeutic agent-targeting agent construct that binds toan aminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, present, expressed, translocated, presented orcomplexed at the luminal surface of blood transporting vessels of thevascularized tumor.

The essence of the invention may also be defined as a compositioncomprising at least a first diagnostic agent-targeting agent construct,or preferably a therapeutic agent-targeting agent construct, preferablythat binds to phosphatidylserine or phosphatidylethanolamine, for use inthe preparation of a medicament for use in tumor vasculature imagingand/or destruction and for human tumor diagnosis and/or treatment. Thiscan also be defined as a composition comprising at least a firstdiagnostic agent-targeting agent construct, or preferably a therapeuticagent-targeting agent construct, for use in the preparation of amedicament for use in binding to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, present, expressed,translocated, presented or complexed at the luminal surface of bloodtransporting vessels of a vascularized tumor and for use in forming animage of tumor vasculature and/or for use in inducing tumor vasculaturedestruction and for human tumor diagnosis and/or treatment.

In the methods, medicaments and uses of the present invention, one ofthe advantages lies in the fact that the provision of the diagnostic ortherapeutic agent-targeting agent construct, preferably one that bindsto phosphatidylserine or phosphatidylethanolamine, into the systemiccirculation of an animal or patient results in the preferential orspecific localization to the tumor vascular surface membranesthemselves, and not to some protein complex more distant from themembrane. The invention thus provides for more intimate cell contactthan the methods and anti-vascular agents of the prior art.

In the context of the present invention, the term “a vascularized tumor”most preferably means a vascularized, malignant tumor, solid tumor or“cancer”. The invention is particularly advantageous in treatingvascularized tumors of at least about intermediate size, and in treatinglarge vascularized tumors—although this is by no means a limitation onthe invention. The invention may therefore be used in the treatment ofany tumor that exhibits aminophospholipid-positive blood vessels,preferably phosphatidylserine- and/or phosphatidylethanolamine-positiveblood vessels.

In preferred embodiments, the tumors to be treated by the presentinvention will exhibit a killing effective number ofaminophospholipid-positive blood vessels. “A killing effective number ofaminophospholipid-positive blood vessels”, as used herein, means that atleast about 3% of the total number of blood vessels within the tumorwill be positive for aminophospholipid expression, preferablyphosphatidylserine and/or phosphatidylethanolamine expression.Preferably, at least about 5%, at least about 8%, or at least about 10%or so, of the total number of blood vessels within the tumor will bepositive for aminophospholipid expression. Given theaminophospholipid-negative, particularly PS-negative, nature of theblood vessels within normal tissues, the tumor vessels will act as sinkfor the administered antibodies. Furthermore, as destruction of only aminimum number of tumor vessels can cause widespread thrombosis,necrosis and an avalanche of tumor cell death, antibody localization toall, or even a majority, of the tumor vessels is not necessary foreffective therapeutic intervention.

Nonetheless, in more preferred embodiments, tumors to be treated by thisinvention will exhibit a significant number ofaminophospholipid-positive blood vessels. “A significant number ofaminophospholipid-positive blood vessels”, as used herein, means that atleast about 10–12% of the total number of blood vessels within the tumorwill be positive for aminophospholipid expression, preferablyphosphatidylserine and/or phosphatidylethanolamine expression. Even morepreferably, the % of aminophospholipid-expressing tumor vessels will beat least about 15%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,or at least about 80% or so of the total number of blood vessels withinthe tumor, up to and including even at least about 90% or 95% of thevessels.

The “therapeutically effective amounts” for use in the invention areamounts of therapeutic agent-targeting agent constructs, preferably PS-or PE-binding constructs, effective to specifically kill at least aportion of tumor or intratumoral vascular endothelial cells; tospecifically promote coagulation in at least a portion of tumor orintratumoral blood vessels; to specifically occlude or destroy at leasta portion of blood transporting vessels of the tumor; to specificallyinduce necrosis in at least a portion of a tumor; and/or to induce tumorregression or remission upon administration to selected animals orpatients. Such effects are achieved while exhibiting little or nobinding to, or little or no killing of, vascular endothelial cells innormal, healthy tissues; little or no coagulation in, occlusion ordestruction of blood vessels in healthy, normal tissues; and exertingnegligible or manageable adverse side effects on normal, healthy tissuesof the animal or patient.

The terms “preferentially” and “specifically”, as used herein in thecontext of promoting coagulation in, or destroying, tumor vasculature,and/or in the context of causing tumor necrosis, thus mean that thetherapeutic agent-targeting agent constructs function to achievecoagulation, destruction and/or tumor necrosis that is substantiallyconfined to the tumor vasculature and tumor site, and does notsubstantially extend to causing coagulation, destruction and/or tissuenecrosis in normal, healthy tissues of the animal or subject. Thestructure and function of healthy cells and tissues is thereforemaintained substantially unimpaired by the practice of the invention.

Although understanding the mechanism of action is not necessary to thepractice of the present invention, the methods will generally operate onthe basis of the mode of action of the particular therapeutic agent oragents chosen for attachment to the targeting agent. As such, theaminophospholipid binding agents that are conjugated to, or operativelyassociated with, cytotoxic or anticellular agents(“anti-aminophospholipid immunotoxins”) will act initially via cellulardestruction. Likewise, aminophospholipid binding agents that areconjugated to, or operatively associated with, coagulation factors(“anti-aminophospholipid coaguligands”) will act initially viacoagulation. However, these mechanisms will have some cross-over, ascell destruction exposes basement membranes and results in coagulation,and as coagulation deprives the cells of oxygen and nutrients andresults in cell destruction.

Naked or unconjugated antibodies against aminophospholipid componentsare also capable of specifically inducing tumor blood vessel destructionand tumor necrosis in vivo. Such methods of tumor treatment are alsocontemplated by the present inventors, and are disclosed and claimed infirst and second provisional application Ser. Nos. 60/092,672 (filedJul. 13, 1998) and 60/110,608 (filed Dec. 2, 1998) and in co-filed U.S.and PCT patent application Ser. Nos. 09/351,543, 09/351,862 andPCT/US99/15600, each specifically incorporated herein by reference. Inlight of the beneficial effects of naked anti-aminophospholipidantibodies, the mechanism of action of the present conjugates may extendbeyond the mode of action of the particular therapeutic agent or agentsemployed.

Therefore, the following mechanisms may contribute to the success of theinvention: cell-mediated cytotoxicity, complement-mediated lysis,apoptosis, antibody-induced cell signaling (direct signaling), ormimicking or altering signal transduction pathways (indirect signaling).

The treatment methods thus include administering to an animal or patienthaving a vascularized tumor at least a first pharmaceutical compositioncomprising an amount of at least a first therapeutic agent-targetingagent construct effective to induce, or specifically induce,cell-mediated cytotoxicity of at least a portion of the tumor orintratumoral vascular endothelial cells. Herein, the first therapeuticagent-targeting agent construct binds to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, present,expressed, translocated, presented or complexed at the luminal surfaceof tumor or intratumoral vascular endothelial cells and inducescell-mediated cytotoxicity of at least a portion of the tumor orintratumoral vascular endothelial cells, as opposed to endothelial cellsin normal vessels. As used herein, “cell-mediated cytotoxicity ordestruction” includes ADCC (antibody-dependent, cell-mediatedcytotoxicity) and NK (natural killer) cell killing.

The methods further include administering to an animal or patient havinga vascularized tumor at least a first pharmaceutical compositioncomprising an amount of at least a first therapeutic agent-targetingagent construct effective to induce, or specifically induce,complement-mediated lysis of at least a portion of the tumor orintratumoral vascular endothelial cells. Herein, the first therapeuticagent-targeting agent construct binds to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, present,expressed, translocated, presented or complexed at the luminal surfaceof tumor or intratumoral vascular endothelial cells and inducescomplement-mediated lysis of at least a portion of the tumor orintratumoral vascular endothelial cells, as opposed to endothelial cellsin normal vessels.

As used herein, “complement-mediated or complement-dependent lysis orcytotoxicity” means the process by which the complement-dependentcoagulation cascade is activated, multi-component complexes areassembled, ultimately generating a lytic complex that has direct lyticaction, causing cell permeabilization. Therapeutic agent-targetingagents for use in inducing complement-mediated lysis will generallyinclude an antibody Fc portion.

The complement-based mechanisms by which the present invention mayoperate further include “complement-activated ADCC”. In such aspects,the administered therapeutic agent-targeting agent contains an antibody,or fragment thereof, that binds to an aminophospholipid, preferablyphosphatidylserine or phosphatidylethanolamine, present, expressed,translocated, presented or complexed at the luminal surface of tumor orintratumoral vascular endothelial cells and induces complement-activatedADCC of at least a portion of the tumor or intratumoral vascularendothelial cells, as opposed to endothelial cells in normal vessels.“Complement-activated ADCC” is used to refer to the process by whichcomplement, not an antibody Fc portion per se, holds a multi-componentcomplex together and in which cells such as neutrophils lyse the targetcell.

In other embodiments, the methods include administering to an animal orpatient having a vascularized tumor at least a first pharmaceuticalcomposition comprising an amount of at least a first therapeuticagent-targeting agent construct effective to induce, or specificallyinduce, apoptosis in at least a portion of the tumor or intratumoralvascular endothelial cells. Herein, the first therapeuticagent-targeting agent construct binds to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, present,expressed, translocated, presented or complexed at the luminal surfaceof tumor or intratumoral vascular endothelial cells and inducesapoptosis in least a portion of the tumor or intratumoral vascularendothelial cells, as opposed to endothelial cells in normal vessels.

As used herein, “induces apoptosis” means induces the process ofprogrammed cell death that, during the initial stages, maintains theintegrity of the cell membrane, yet transmits the death-inducing signalsinto the cell. This is opposed to the mechanisms of cell necrosis,during which the cell membrane loses its integrity and becomes leaky atthe onset of the process.

Therapeutic benefits may be realized by the administration of at leasttwo, three or more therapeutic agent-targeting agent constructs. Thetherapeutic agent-targeting agent constructs may also be combined withother therapies to provide combined therapeutically effective amounts,as disclosed herein.

The treatment methods of the present invention will generally involvethe administration of the pharmaceutically effective composition to theanimal systemically, such as via intravenous injection. However, anyroute of administration that allows the therapeutic agent-targetingagent construct to localize to the tumor or intratumoral vascularendothelial cells will be acceptable.

“Administration”, as used herein, therefore means provision or deliveryof therapeutic agent-targeting agent constructs in an amount(s) and fora period of time(s) effective to allow binding to an aminophospholipid,preferably phosphatidylserine or phosphatidylethanolamine, present,expressed, translocated, presented or complexed at the luminal surfaceof blood transporting vessels of the vascularized tumor, and to exert atumor vasculature destructive and tumor-regressive effect. The passiveadministration of proteinaceous therapeutic agent-targeting agentconstructs is generally preferred, in part, for its simplicity andreproducibility.

However, the term “administration” is herein used to refer to any andall means by which therapeutic agent-targeting agent constructs aredelivered or otherwise provided to the tumor vasculature.“Administration” therefore includes the provision of cells that producethe therapeutic agent-targeting agent constructs in a manner effectiveto result in the delivery of the therapeutic agent-targeting agentconstructs to the tumor vasculature, and/or their localization to suchvasculature. In such embodiments, it may be desirable to formulate orpackage the cells in a selectively permeable membrane, structure orimplantable device, generally one that can be removed to cease therapy.Exogenous therapeutic agent-targeting agent administration will stillgenerally be preferred, as this represents a non-invasive method thatallows the dose to be closely monitored and controlled.

The “therapeutic agent-targeting agent administration methods” of theinvention also extend to the provision of nucleic acids that encodetherapeutic agent-targeting agent constructs in a manner effective toresult in the expression of the therapeutic agent-targeting agentconstructs in the vicinity of the tumor vasculature, and/or in theexpression of therapeutic agent-targeting agent constructs that canlocalize to the tumor vasculature. Any gene therapy technique may beemployed, such as naked DNA delivery, recombinant genes and vectors,cell-based delivery, including ex vivo manipulation of patients' cells,and the like.

One of the benefits of the present invention is that aminophospholipids,particularly phosphatidylserine and phosphatidylethanolamine, aregenerally expressed or available throughout the tumor vessels.Aminophospholipid expression on established, intratumoral blood vesselsis advantageous as targeting and destroying such vessels will rapidlylead to anti-tumor effects. However, so long as the administeredtherapeutic agent-targeting agent constructs bind to at least a portionof the blood transporting vessels, significant anti-tumor effects willensue. This will not be problematical as aminophospholipids, such asphosphatidylserine and phosphatidylethanolamine, are expressed on thelarge, central vessels, and also on veins, venules, arteries, arteriolesand blood transporting capillaries in all regions of the tumor.

In any event, the ability of the therapeutic agent-targeting agentconstructs to destroy the tumor vasculature means that tumor regressioncan be achieved, rather than only tumor stasis. Tumor stasis is mostoften the result of anti-angiogenic therapies that target only thebudding vessels at the periphery of a solid tumor and stop the vesselsproliferating. Even if the present invention targeted more of theperipheral regions of the tumor in certain tumor types, which is notcurrently believed to be the case, destruction of the blood transportingvessels in such areas would still lead to widespread thrombosis andtumor necrosis.

The targeting portions of the diagnostic and/or therapeuticagent-targeting agent constructs of the present invention, whetherbinding to phosphatidylethanolamine or phosphatidylserine, may be eitherantibody-based or binding ligand or binding protein based. Anyaminophospholipid binding ligand or protein known in the art may thusnow be advantageously used in the delivery of therapeutic agents totumor vasculature.

By way of example only, suitable aminophospholipid binding ligands andproteins include low and high molecular weight kininogens and other rat,bovine, monkey or human phosphatidylethanolamine binding proteins; andany one or more of a number of phosphatidylserine-serine bindingannexins. The protein and DNA sequences for such binding ligands areknown in the art and incorporated herein by reference, facilitating theproduction of recombinant fusion proteins for use in the presentinvention.

Aminophospholipid binding reagents encompassed by the term“aminophospholipid binding ligands or binding proteins” extend to allaminophospholipid binding ligands and proteins from all species, andaminophospholipid binding fragments thereof, including dimeric, trimericand multimeric ligands and proteins; bispecific ligands and proteins;chimeric ligands and proteins; human ligands and proteins; recombinantand engineered ligands and proteins, and fragments thereof.

Where antibody-based targeting portions are employed, whether binding tophosphatidylethanolamine or phosphatidylserine, the term“anti-aminophospholipid antibody”, as used herein, refers broadly to anyimmunologic binding agent, such as polyclonal and monoclonal IgG, IgM,IgA, IgD and IgE antibodies. Generally, IgG and/or IgM are preferredbecause they are the most common antibodies in the physiologicalsituation and because they are most easily made in a laboratory setting.

Polyclonal anti-aminophospholipid antibodies, obtained from antisera,may be employed in the invention. However, the use of monoclonalanti-aminophospholipid antibodies (MAbs) will generally be preferred.MAbs are recognized to have certain advantages, e.g., reproducibilityand large-scale production, that makes them suitable for clinicaltreatment. The invention thus provides monoclonal antibodies of themurine, human, monkey, rat, hamster, rabbit and even frog or chickenorigin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will be used in certainembodiments.

As will be understood by those in the art, the immunologic bindingreagents encompassed by the term “anti-aminophospholipid antibody”extend to all anti-aminophospholipid antibodies from all species, andantigen binding fragments thereof, including dimeric, trimeric andmultimeric antibodies; bispecific antibodies; chimeric antibodies; humanand humanized antibodies; recombinant and engineered antibodies, andfragments thereof.

The term “anti-aminophospholipid antibody” is thus used to refer to anyanti-aminophospholipid antibody-like molecule that has an antigenbinding region, and includes antibody fragments such as Fab′, Fab,F(ab′)₂, single domain antibodies (DABs), Fv, scFv (single chain Fv),and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art.

In certain embodiments, the antibodies employed in the therapeuticagent-targeting agent constructs will be “humanized” or humanantibodies. “Humanized” antibodies are generally chimeric monoclonalantibodies from mouse, rat, or other non-human species, bearing humanconstant and/or variable region domains (“part-human chimericantibodies”). Mostly, humanized monoclonal antibodies for use in thepresent invention will be chimeric antibodies wherein at least a firstantigen binding region, or complementarity determining region (CDR), ofa mouse, rat or other non-human anti-aminophospholipid monoclonalantibody is operatively attached to, or “grafted” onto, a human antibodyconstant region or “framework”.

“Humanized” monoclonal antibodies for use herein may also beanti-aminophospholipid monoclonal antibodies from non-human specieswherein one or more selected amino acids have been exchanged for aminoacids more commonly observed in human antibodies. This can be readilyachieved through the use of routine recombinant technology, particularlysite-specific mutagenesis.

Entirely human, rather than “humanized”, anti-aminophospholipidantibodies may also be prepared and used in the therapeuticagent-targeting agent constructs of the present invention. Such humanantibodies may be polyclonal antibodies, as obtained from human patientsthat have any one or more of a variety of diseases, disorders orclinical conditions associated with the production ofanti-aminophospholipid antibodies. Such antibodies may be concentrated,partially purified or substantially purified for use herein.

A range of techniques are also available for preparing human monoclonalantibodies. As human patients with anti-aminophospholipidantibody-producing diseases exist, the anti-aminophospholipidantibody-producing cells from such patients may be obtained andmanipulated in vitro to provide a human monoclonal antibody for use in atherapeutic agent-targeting agent construct. The in vitro manipulationsor techniques include fusing to prepare a monoclonal antibody-producinghybridoma, and/or cloning the gene(s) encoding theanti-aminophospholipid antibody from the cells (“recombinant humanantibodies”).

Human anti-aminophospholipid antibody-producing cells may also beobtained from human subjects without an anti-aminophospholipidantibody-associated disease, i.e. “healthy subjects” in the context ofthe present invention. To achieve this, one would simply obtain apopulation of mixed peripheral blood lymphocytes from a human subject,including antigen-presenting and antibody-producing cells, and stimulatethe cell population in vitro by admixing with an immunogenicallyeffective amount of an aminophospholipid sample. Again, the humananti-aminophospholipid antibody-producing cells, once obtained, could beused in hybridoma and/or recombinant antibody production prior totherapeutic agent-targeting agent construct preparation.

Further techniques for human monoclonal antibody production includeimmunizing a transgenic animal, preferably a transgenic mouse, thatcomprises a human antibody library with an immunogenically effectiveamount of an aminophospholipid sample. This also generates humananti-aminophospholipid antibody-producing cells for further manipulationin hybridoma and/or recombinant antibody production, with the advantagethat spleen cells, rather than peripheral blood cells, can be readilyobtained from the transgenic animal or mouse.

Preferred anti-aminophospholipid antibodies for use in the therapeuticagent-targeting agent constructs of the present invention areanti-phosphatidylserine (anti-PS) and anti-phosphatidylethanolamine(anti-PE) antibodies. Anti-PS antibodies will generally recognize, bindto or have immunospecificity for the PS molecule present, expressed,translocated, presented or complexed at the luminal surface of tumorvascular endothelial cells. Suitable antibodies will thus bind tophosphatidyl-L-serine (Umeda et al., 1989; incorporated herein byreference). Anti-PE antibodies will generally recognize, bind to or haveimmunospecificity for the PE molecule present, expressed, translocated,presented or complexed at the luminal surface of tumor vascularendothelial cells.

Administering diagnostic and/or therapeutic agent-targeting agentconstructs to an animal with a tumor will result in specific binding tothe aminophospholipid molecules present, expressed or translocated tothe luminal surface of the tumor blood vessels, i.e., the therapeuticagent-targeting agent constructs will bind to the aminophospholipidmolecules in a natural, biological environment. Therefore, no particularmanipulation will be necessary to ensure binding.

However, in terms of antibody binding, it is of scientific interest tonote that aminophospholipids may be most frequently recognized, orbound, by anti-aminophospholipid antibodies when the aminophospholipidmolecules are associated with one or more proteins or other non-lipidbiological components. For example, anti-PS antibodies that occur as asub-set of anti-phospholipid (anti-PL) antibodies in patients withcertain diseases and disorders are now believed to bind to PS incombination with proteins such as β₂-glycoprotein I (β2-GPI orapolipoprotein H, apoH) and prothrombin (U.S. Pat. No. 5,344,758; Rote,1996; each incorporated herein by reference). Similarly, anti-PEantibodies that occur in disease states are now believed to bind to PEin combination with proteins such as low and high molecular weightkininogen (HK), prekallikrein and even factor XI (Sugi and McIntyre,1995; 1996a; 1996b; each incorporated herein by reference).

This is the meaning of the terms “presented” and “complexed at” theluminal surface of tumor blood vessels, as used herein, which mean thatthe aminophospholipid molecules are present at the surface of tumorblood vessels in a binding competent state, or antibody-bindingcompetent state, irrespective of the molecular definition of thatparticular state. PS may even be targeted as a complex with factorII/IIa, VII/VIIa, IX/IXa and X/Xa. Moreover, the nature of theaminophospholipid target may change during practice of the invention, asthe initial aminophospholipid antibody binding, anti-endothelial celland anti-tumor effects may result in biological changes that alter thenumber, conformation and/or type of the aminophospholipid targetepitope(s).

The term “anti-aminophospholipid antibody”, as used in the context ofthe present invention, therefore means any antibody, immunologicalbinding agent or antisera; monoclonal, human, humanized, dimeric,trimeric, multimeric, chimeric, bispecific, recombinant or engineeredantibody; or Fab′, Fab, F(ab′)₂, DABs, Fv or scFv antigen bindingfragment thereof; that at least binds to a lipid and aminogroup-containing complex or aminophospholipid target, preferably aphosphatidylserine- or phosphatidylethanolamine-based target.

The requirement that the antibody “at least bind to an aminophospholipidtarget” is met by the antibody binding to any and/or all physiologicallyrelevant forms of aminophospholipids, including so-called “hexagonal”and “hexagonal phase II” PS and PE (HexII PS and HexII PE) (Rauch etal., 1986; Rauch and Janoff, 1990; Berard et al., 1993; eachincorporated herein by reference) and PS and PE in combination with anyother protein, lipid, membrane component, plasma or serum component, orany combination thereof. Thus, an “anti-aminophospholipid antibody” isan antibody that binds to an aminophospholipid in the tumor bloodvessels, notwithstanding the fact that bilayer or micelleaminophospholipids may be considered to be immunogenically neutral.

The anti-aminophospholipid antibodies may recognize, bind to or haveimmunospecificity for aminophospholipid molecules, or an immunogeniccomplex thereof (including hexagonal aminophospholipids and proteincombinations), to the exclusion of other phospholipids or lipids. Suchantibodies may be termed “aminophospholipid-specific oraminophospholipid-restricted antibodies”, and their use in thetherapeutic agent-targeting agent constructs of the invention will oftenbe preferred. “Aminophospholipid-specific oraminophospholipid-restricted antibodies” will generally exhibitsignificant binding to aminophospholipids, while exhibiting little or nosignificant binding to other lipid components, such asphosphatidylinositol (PI), phosphatidylglycerol (PG) and evenphosphatidylcholine (PC) in certain embodiments.

“PS-specific or PS-restricted antibodies” will generally exhibitsignificant binding to PS, while exhibiting little or no significantbinding to lipid components such as phosphatidylethanolamine andcardiolipin (CL), as well as PC, PI and PG. “PE-specific orPE-restricted antibodies” will generally exhibit significant binding toPE, while exhibiting little or no significant binding to lipidcomponents such as phosphatidylserine and cardiolipin, as well as PC, PIand PG. The preparation of specific anti-aminophospholipid antibodies isreadily achieved, e.g., as disclosed by Rauch et al. (1986); Umeda etal. (1989); Rauch and Janoff (1990); and Rote et al. (1993); eachincorporated herein by reference.

“Cross-reactive anti-aminophospholipid antibodies” that recognize, bindto or have immunospecificity for an aminophospholipid molecule, or animmunogenic complex thereof (including hexagonal aminophospholipids andprotein combinations), in addition to exhibiting lesser but detectablebinding to other phospholipid or lipid components are by no meansexcluded from use in the invention. Such “cross-reactiveanti-aminophospholipid antibodies” may be employed so long as they bindto an aminophospholipid present, expressed, translocated, presented orcomplexed at the luminal surface of tumor vascular endothelial cells invivo.

Further suitable aminophospholipid-specific oraminophospholipid-restricted antibodies are those anti-aminophospholipidantibodies that bind to both PS and PE. While clearly being specific orrestricted to aminophospholipids, as opposed to other lipid components,antibodies exist that bind to each of the preferred targets of thepresent invention. Examples of such antibodies for use in thetherapeutic agent-targeting agent constructs of the invention include,but are not limited to, PS3A, PSF6, PSF7, PSB4, PS3H1 and PS3E10(Igarashi et al., 1991; incorporated herein by reference)

Further exemplary anti-PS antibodies for use in the therapeuticagent-targeting agent constructs include, but are not limited toBA3B5C4, PS4A7, PS1G3 and 3SB9b; with PS4A7, PS1G3 and 3SB9b generallybeing preferred. Monoclonal antibodies, humanized antibodies and/orantigen-binding fragments based upon the 3SB9b antibody (Rote et al.,1993; incorporated herein by reference) are currently most preferred.

Although aminophospholipids, such as PS and PE, in bilayer or micelleform have been reported to be non- or weakly antigenic, or non- orweakly-immunogenic, the scientific literature has reported nodifficulties in generating anti-aminophospholipid antibodies, such asanti-PS and anti-PE antibodies. Anti-aminophospholipid antibodies ormonoclonal antibodies may therefore be readily prepared by preparativeprocesses and methods that comprise:

-   -   (a) preparing an anti-aminophospholipid antibody-producing cell;        and    -   (b) obtaining an anti-aminophospholipid antibody or monoclonal        antibody from the antibody-producing cell.

The processes of preparing anti-aminophospholipid antibody-producingcells and obtaining anti-aminophospholipid antibodies therefrom may beconduced in situ in a given patient. That is, simply providing animmunogenically effective amount of an immunogenic aminophospholipidsample to a patient will result in anti-aminophospholipid antibodygeneration. Thus, the anti-aminophospholipid antibody is still“obtained” from the antibody-producing cell, but it does not have to beisolated away from a host and subsequently provided to a patient, beingable to spontaneously localize to the tumor vasculature and exert itsbiological anti-tumor effects.

As disclosed herein, anti-aminophospholipid antibody-producing cells maybe obtained, and antibodies subsequently isolated and/or purified, fromhuman patients with anti-aminophospholipid antibody-producing diseases,from stimulating peripheral blood lymphocytes with aminophospholipids invitro, and also by immunization processes and methods. The latter ofwhich generally comprise:

-   -   (a) immunizing an animal by administering to the animal at least        one dose, and optionally more than one dose, of an        immunogenically effective amount of an immunogenic        aminophospholipid sample (such as a hexagonal, or hexagonal        phase II form of an aminophospholipid), preferably an        immunogenic PS or PE sample; and    -   (b) obtaining an anti-aminophospholipid antibody-producing cell        from the immunized animal.

The immunogenically effective amount of the aminophospholipid sample orsamples may be a Salmonella-coated aminophospholipid sample (Umeda etal., 1989; incorporated herein by reference); an aminophospholipidmicelle, liposome, lipid complex or lipid formulation sample; or anaminophospholipid sample fabricated with SDS. Any such aminophospholipidsample may be administered in combination with any suitable adjuvant,such as Freund's complete adjuvant (Rote et al., 1993; incorporatedherein by reference). Any empirical technique or variation may beemployed to increase immunogenicity, and/or hexagonal or hexagonal phaseII forms of the aminophospholipids may be administered.

The immunization may be based upon one or more intrasplenic injectionsof an immunogenically effective amount of an aminophospholipid sample(Umeda et al., 1989; incorporated herein by reference).

Irrespective of the nature of the immunization process, or the type ofimmunized animal, anti-aminophospholipid antibody-producing cells areobtained from the immunized animal and, preferably, further manipulatedby the hand of man. “An immunized animal”, as used herein, is anon-human animal, unless otherwise expressly stated. Although anyantibody-producing cell may be used, most preferably, spleen cells areobtained as the source of the antibody-producing cells. Theanti-aminophospholipid antibody-producing cells may be used in apreparative process that comprises:

-   -   (a) fusing an anti-aminophospholipid antibody-producing cell        with an immortal cell to prepare a hybridoma that produces an        anti-aminophospholipid monoclonal antibody and    -   (b) obtaining an anti-aminophospholipid monoclonal antibody from        the hybridoma.

Hybridoma-based monoclonal antibody preparative methods thus includethose that comprise:

-   -   (a) immunizing an animal by administering to the animal at least        one dose, and optionally more than one dose, of an        immunogenically effective amount of an immunogenic        aminophospholipid sample (such as a hexagonal, or hexagonal        phase II form of an aminophospholipid), preferably an        immunogenic PS or PE sample;    -   (b) preparing a collection of monoclonal antibody-producing        hybridomas from the immunized animal;    -   (c) selecting from the collection at least a first hybridoma        that produces at least a first anti-aminophospholipid monoclonal        antibody, and preferably, at least a first        aminophospholipid-specific monoclonal antibody; and    -   (d) culturing the at least a first        anti-aminophospholipid-producing or aminophospholipid-specific        hybridoma to provide the at least a first anti-aminophospholipid        monoclonal antibody or aminophospholipid-specific monoclonal        antibody; and preferably    -   (e) obtaining the at least a first anti-aminophospholipid        monoclonal antibody or aminophospholipid-specific monoclonal        antibody from the cultured at least a first hybridoma.

As non-human animals are used for immunization, theanti-aminophospholipid monoclonal antibodies obtained from such ahybridoma will often have a non-human make up. Such antibodies may beoptionally subjected to a humanization process, grafting or mutation, asknown to those of skill in the art and further disclosed herein.Alternatively, transgenic animals, such as mice, may be used thatcomprise a human antibody gene library. Immunization of such animalswill therefore directly result in the generation of humananti-aminophospholipid antibodies.

After the production of a suitable antibody-producing cell, mostpreferably a hybridoma, whether producing human or non-human antibodies,the monoclonal antibody-encoding nucleic acids may be cloned to preparea “recombinant” monoclonal antibody. Any recombinant cloning techniquemay be utilized, including the use of PCR to prime the synthesis of theantibody-encoding nucleic acid sequences. Therefore, yet furtherappropriate monoclonal antibody preparative methods include those thatcomprise using the anti-aminophospholipid antibody-producing cells asfollows:

-   -   (a) obtaining at least a first anti-aminophospholipid        antibody-encoding nucleic acid molecule or segment from an        anti-aminophospholipid antibody-producing cell, preferably a        hybridoma; and    -   (b) expressing the nucleic acid molecule or segment in a        recombinant host cell to obtain a recombinant        anti-aminophospholipid monoclonal antibody.

However, other powerful recombinant techniques are available that areideally suited to the preparation of recombinant monoclonal antibodies.Such recombinant techniques include the phagemid library-basedmonoclonal antibody preparative methods comprising:

-   -   (a) immunizing an animal by administering to the animal at least        one dose, and optionally more than one dose, of an        immunogenically effective amount of an immunogenic        aminophospholipid sample (such as a hexagonal, or hexagonal        phase II form of an aminophospholipid), preferably an        immunogenic PS or PE sample;    -   (b) preparing a combinatorial immunoglobulin phagemid library        expressing RNA isolated from the antibody-producing cells,        preferably from the spleen, of the immunized animal;    -   (c) selecting from the phagemid library at least a first clone        that expresses at least a first anti-aminophospholipid antibody,        and preferably, at least a first aminophospholipid-specific        antibody;    -   (d) obtaining anti-aminophospholipid antibody-encoding nucleic        acids from the at least a first selected clone and expressing        the nucleic acids in a recombinant host cell to provide the at        least a first anti-aminophospholipid antibody or        aminophospholipid-specific antibody; and preferably    -   (e) obtaining the at least a first anti-aminophospholipid        antibody or aminophospholipid-specific antibody expressed by the        nucleic acids obtained from the at least a first selected clone.

Again, in such phagemid library-based techniques, transgenic animalsbearing human antibody gene libraries may be employed, thus yieldingrecombinant human monoclonal antibodies.

Irrespective of the manner of preparation of a firstanti-aminophospholipid antibody nucleic acid segment, further suitableanti-aminophospholipid antibody nucleic acid segments may be readilyprepared by standard molecular biological techniques. In order toconfirm that any variant, mutant or second generationanti-aminophospholipid antibody nucleic acid segment is suitable for usein the present invention, the nucleic acid segment will be tested toconfirm expression of an antibody that binds to an aminophospholipid.Preferably, the variant, mutant or second generationanti-aminophospholipid antibody nucleic acid segment will also be testedto confirm hybridization to an anti-aminophospholipid antibody nucleicacid segment under standard, more preferably, standard stringenthybridization conditions. Exemplary suitable hybridization conditionsinclude hybridization in about 7% sodium dodecyl sulfate (SDS), about0.5 M NaPO₄, about 1 mM EDTA at about 50° C.; and washing with about 1%SDS at about 42° C.

As a variety of recombinant monoclonal antibodies, whether human ornon-human in origin, may be readily prepared, the treatment methods ofthe invention may be executed by providing to the animal or patient atleast a first nucleic acid segment that expresses a biologicallyeffective amount of at least a first therapeutic agent-targeting agentconstruct in the patient. The “nucleic acid segment that expresses atherapeutic agent-targeting agent construct” will generally be in theform of at least an expression construct, and may be in the form of anexpression construct comprised within a virus or within a recombinanthost cell. Preferred gene therapy vectors of the present invention willgenerally be viral vectors, such as comprised within a recombinantretrovirus, herpes simplex virus (HSV), adenovirus, adeno-associatedvirus (AAV), cytomegalovirus (CMV), and the like.

Once a targeting agent has been selected, whether antibody-based orbinding ligand-based, and whether binding tophosphatidylethanolamineand/or phosphatidylserine, the targeting agentis operatively attached to one or more diagnostic and/or therapeuticagents or “effector” portions. The therapeutic agents of the presentconstructs will generally be either anti-cellular, cytotoxic oranti-angiogenic agents, or coagulation factors (coagulants).

The use of anti-cellular, cytotoxic and/or anti-angiogenic agentsresults in “aminophospholipid immunotoxins” (or anti-aminophospholipidimmunotoxins), whereas the use of coagulation factors results in“aminophospholipid coaguligands” (or anti-aminophospholipidcoaguligands). These terms are again used for simplicity and succinctlyrefer to aminophospholipid binding ligands or therapeuticagent-aminophospholipid targeting agent constructs in terms of theirattached therapeutic moiety.

The present invention further provides binding ligands, and methods ofuse, comprising at least two therapeutic agents operatively attached toa targeting agent comprising a single aminophospholipid binding site.The binding ligands may comprise at least two therapeutic agentsoperatively attached to a targeting agent that comprises at least twoaminophospholipid binding sites; or a plurality of therapeutic agentsoperatively attached to a targeting agent that comprises a plurality ofaminophospholipid binding sites, generally at regions distinct from theaminophospholipid binding sites.

Combinations of anti-cellular and cytotoxic agents with coagulationfactors are also contemplated, irrespective of the number ofaminophospholipid binding sites. The combined use of therapeutic agentsof different classes, such as cytotoxins and coagulants, is alsocontemplated in embodiments where two or more binding ligands areadministered to the animal, each containing a single type of therapeuticagent. Different cytotoxins may also be employed in one or more bindingligands or methods, such as DNA synthesis inhibitors combined withclassic cytotoxins, such as ricin.

In certain applications, the aminophospholipid-targeted constructs willbe operatively attached to cytotoxic, cytostatic or otherwiseanti-cellular agents that have the ability to kill or suppress thegrowth or cell division of endothelial cells. Suitable anti-cellularagents include chemotherapeutic agents, as well as cytotoxins andcytostatic agents. Cytostatic agents are generally those that disturbthe natural cell cycle of a target cell, preferably so that the cell istaken out of the cell cycle.

Exemplary chemotherapeutic agents include: steroids; cytokines;anti-metabolites, such as cytosine arabinoside, fluorouracil,methotrexate or aminopterin; anthracyclines; mitomycin C; vincaalkaloids; antibiotics; demecolcine; etoposide; mithramycin; andanti-tumor alkylating agents, such as chlorambucil or melphalan. Indeed,any of the agents disclosed herein in Table C could be used. Certainpreferred anti-cellular agents are DNA synthesis inhibitors, such asdaunorubicin, doxorubicin, adriamycin, and the like.

In other embodiments, aminophospholipid-targeted constructs of theinvention may be operatively attached to anti-angiogenic agents that,acting either alone or in concert with other host factors, oradministered therapeutic agents, have the ability to prevent or inhibitvascularization and/or to induce regression of blood vessels. Suitableanti-angiogenic agents include those listed in Table D, as well as otheranti-angiogenic agents known to those of skill in the art. By way ofexample only, one may mention the angiopoietins, preferably,angiopoietin-2 (Ang-2; SEQ ID NO:3 and SEQ ID NO:4), but alsoangiopoietin-1 (Ang-1; SEQ ID NO:1 and SEQ ID NO:2), angiopoietin fusionproteins (for example, as in SEQ ID NO:5), and even angiopoietin-3 andangiopoietin-4.

In certain therapeutic applications, toxin moieties will be preferred,due to the much greater ability of most toxins to deliver a cell killingeffect, as compared to other potential agents. Therefore, certainpreferred anti-cellular agents for aminophospholipid-targeted constructsare plant-, fungus- or bacteria-derived toxins. Exemplary toxins includeepipodophyllotoxins; bacterial endotoxin or the lipid A moiety ofbacterial endotoxin; ribosome inactivating proteins, such as saporin orgelonin; α-sarcin; aspergillin; restrictocin; ribonucleases, such asplacental ribonuclease; diphtheria toxin and pseudomonas exotoxin.

Preferred toxins for certain embodiments are gelonin and/or the A chaintoxins, such as ricin A chain. The most preferred toxin moiety is oftenricin A chain that has been treated to modify or remove carbohydrateresidues, so called “deglycosylated A chain” (dgA). Deglycosylated ricinA chain is preferred because of its extreme potency, longer half-life,and because it is economically feasible to manufacture it a clinicalgrade and scale. Recombinant and/or truncated ricin A chain may also beused.

For tumor targeting and treatment with immunotoxins, the followingpatents and patent applications are specifically incorporated herein byreference for the purposes of even further supplementing the presentteachings regarding anticellular and cytotoxic agents: U.S. Pat. Nos.5,855,866; 5,776,427; 5,863,538; 6,004,554; 5,965,132; 6,051,230 and5,660,827; and U.S. application Ser. No. 07/846,349.

The aminophospholipid-targeted constructs of the invention may comprisea component that is capable of promoting coagulation, i.e., a coagulant.Here, the targeting antibody or ligand may be directly or indirectly,e.g., via another antibody, linked to a factor that directly orindirectly stimulates coagulation.

Preferred coagulation factors for such uses are Tissue Factor (TF) andTF derivatives, such as truncated TF (tTF), dimeric, trimeric,polymeric/multimeric TF, and mutant TF deficient in the ability toactivate Factor VII. Other suitable coagulation factors include vitaminK-dependent coagulants, such as Factor II/IIa, Factor VII/VIIa, FactorIX/IXa and Factor X/Xa; vitamin K-dependent coagulation factors thatlack the G1a modification; Russell's viper venom Factor X activator;platelet-activating compounds, such as thromboxane A₂ and thromboxane A₂synthase; and inhibitors of fibrinolysis, such as α2-antiplasmin.

Tumor targeting and treatment with coaguligands is described in thefollowing patents and patent applications, each of which arespecifically incorporated herein by reference for the purposes of evenfurther supplementing the present teachings regarding coaguligands andcoagulation factors: U.S. Pat. Nos. 5,855,866; 5,965,132; 6,036,955;5,877,289; and 6,093,399; and application Ser. No. 07/846,349.

As somewhat wider distribution of a coagulating agent will not beassociated with severe side effects, there is a less stringentrequirement imposed on the targeting element of coaguligands than withimmunotoxins. Therefore, to achieve specific targeting means thatcoagulation is promoted in the tumor vasculature relative to thevasculature in non-tumor sites. Thus, specific targeting of acoaguligand is a functional term, rather than a purely physical termrelating to the biodistribution properties of the targeting agent.

The preparation of immunotoxins is generally well known in the art (see,e.g., U.S. Pat. No. 4,340,535, incorporated herein by reference). Eachof the following patents and patent applications are furtherincorporated herein by reference for the purposes of even furthersupplementing the present teachings regarding immunotoxin generation,purification and use: U.S. Pat. Nos. 5,855,866; 5,776,427; 5,863,538;6,004,554; 5,965,132; 6,051,230; 5,660,827; and U.S. application Ser.No. 07/846,349.

In the preparation of immunotoxins, advantages may be achieved throughthe use of certain linkers. For example, linkers that contain adisulfide bond that is sterically “hindered” are often preferred, due totheir greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action. It is generally desiredto have a conjugate that will remain intact under conditions foundeverywhere in the body except the intended site of action, at whichpoint it is desirable that the conjugate have good “release”characteristics.

Depending on the specific toxin compound used, it may be necessary toprovide a peptide spacer operatively attaching the targeting agent andthe toxin compound, wherein the peptide spacer is capable of foldinginto a disulfide-bonded loop structure. Proteolytic cleavage within theloop would then yield a heterodimeric polypeptide wherein the targetingagent and the toxin compound are linked by only a single disulfide bond.

When certain other toxin compounds are utilized, a non-cleavable peptidespacer may be provided to operatively attach the targeting agent and thetoxin compound. Toxins that may be used in conjunction withnon-cleavable peptide spacers are those that may, themselves, beconverted by proteolytic cleavage, into a cytotoxic disulfide-bondedform. An example of such a toxin compound is a Pseudonomas exotoxincompound.

A variety of chemotherapeutic and other pharmacological agents can alsobe successfully conjugated to aminophospholipid antibodies or targetingligands. Exemplary antineoplastic agents that have been conjugated toantibodies include doxorubicin, daunomycin, methotrexate andvinblastine. Moreover, the attachment of other agents such asneocarzinostatin, macromycin, trenimon and α-amanitin has been described(see U.S. Pat. No. 5,855,866 and 5,865,132 STET therein.

In light of one of the present inventors earlier work, the preparationof coaguligands is now also easily practiced. The operable associationof one or more coagulation factors with an aminophospholipid targetingagent may be a direct linkage, such as those described above for theimmunotoxins. Alternatively, the operative association may be anindirect attachment, such as where the targeting agent is operativelyattached to a second binding region, preferably and antibody or antigenbinding region of an antibody, that binds to the coagulation factor. Thecoagulation factor should be attached to the targeting agent at a sitedistinct from its functional coagulating site, particularly where acovalent linkage is used to join the molecules.

Indirectly linked coaguligands are often based upon bispecificantibodies. The preparation of bispecific antibodies is also well knownin the art. One preparative method involves the separate preparation ofantibodies having specificity for the targeted tumor component, on theone hand, and the coagulating agent on the other. Peptic F(ab′γ)₂fragments from the two chosen antibodies are then generated, followed byreduction of each to provide separate Fab′γSH fragments. The SH groupson one of the two partners to be coupled are then alkylated with across-linking reagent, such as o-phenylenedimaleimide, to provide freemaleimide groups on one partner. This partner may then be conjugated tothe other by means of a thioether linkage, to give the desired F(ab′γ)₂heteroconjugate (Glennie et al., 1987; incorporated herein byreference). Other approaches, such as cross-linking with SPDP or proteinA may also be carried out.

Another method for producing bispecific antibodies is by the fusion oftwo hybridomas to form a quadroma. As used herein, the term “quadroma”is used to describe the productive fusion of two B cell hybridomas.Using now standard techniques, two antibody producing hybridomas arefused to give daughter cells, and those cells that have maintained theexpression of both sets of clonotype immunoglobulin genes are thenselected.

A preferred method of generating a quadroma involves the selection of anenzyme deficient mutant of at least one of the parental hybridomas. Thisfirst mutant hybridoma cell line is then fused to cells of a secondhybridoma that had been lethally exposed, e.g., to iodoacetamide,precluding its continued survival. Cell fusion allows for the rescue ofthe first hybridoma by acquiring the gene for its enzyme deficiency fromthe lethally treated hybridoma, and the rescue of the second hybridomathrough fusion to the first hybridoma. Preferred, but not required, isthe fusion of immunoglobulins of the same isotype, but of a differentsubclass. A mixed subclass antibody permits the use if an alternativeassay for the isolation of a preferred quadroma.

Microtiter identification embodiments, FACS, immunofluorescencestaining, idiotype specific antibodies, antigen binding competitionassays, and other methods common in the art of antibody characterizationmay be used to identify preferred quadromas. Following the isolation ofthe quadroma, the bispecific antibodies are purified away from othercell products. This may be accomplished by a variety of antibodyisolation procedures, known to those skilled in the art ofimmunoglobulin purification (see, e.g., Antibodies: A Laboratory Manual,1988; incorporated herein by reference). Protein A or protein Gsepharose columns are preferred.

In the preparation of both immunotoxins and coaguligands, recombinantexpression may be employed. The nucleic acid sequences encoding thechosen targeting agent, and toxin or coagulant, are attached in-frame inan expression vector. Recombinant expression thus results in translationof the nucleic acid to yield the desired targeting agent-toxin/coagulantcompound.

Chemical cross-linkers and avidin:biotin bridges may also join thetherapeutic agent(s) to the targeting agent(s).

The following patents and patent applications are each incorporatedherein by reference for the purposes of even further supplementing thepresent teachings regarding coaguligand preparation, purification anduse, including bispecific antibody coaguligands: U.S. Pat. Nos.5,855,866; 5,965,132; 6,004,555; 6,036,955; 5,877,289; and 6,093,399;U.S. applications Ser. Nos. 07,846,349; 08/273,567; 08/485,482,08/472,631; and 08/481,904.

In certain embodiments, the vasculature of the vascularized tumor of theanimal or patient to be treated may be first imaged. Generally this isachieved by first administering to the animal or patient adiagnostically effective amount of at least a first pharmaceuticalcomposition comprising at least a first detectably-labeledaminophospholipid binding construct that binds to and identifies anaminophospholipid, preferably phosphatidylserine orphosphatidylethanolamine, present, expressed, translocated, presented orcomplexed at the luminal surface of blood vessels or intratumoral bloodvessels of the vascularized tumor. The invention thus further providescompositions for use in, and methods of, distinguishing between tumorand/or intratumoral blood vessels and normal blood vessels. The“distinguishing” is achieved by administering one or more of thedetectably-labeled aminophospholipid binding constructs described.

The detectably-labeled aminophospholipid binding construct may comprisean X-ray detectable compound, such as bismuth (III), gold (III),lanthanum (III) or lead (II); a radioactive ion, such as copper⁶⁷,gallium⁶⁷, gallium⁶⁸, indium¹¹¹, indium¹¹³, iodine¹²³, iodine¹²⁵,iodine¹³¹, mercury¹⁹⁷, mercury²⁰³, rhenium¹⁸⁶, rhenium¹⁸⁸, rubidium⁹⁷,rubidium¹⁰³, technetium^(99m) or yttrium⁹⁰; a nuclear magneticspin-resonance isotope, such as cobalt (II), copper (II), chromium(III), dysprosium (III), erbium (III), gadolinium (III), holmium (III),iron (II), iron (III), manganese (II), neodymium (III), nickel (II),samarium (III), terbium (III), vanadium (II) or ytterbium (III); orrhodamine or fluorescein.

Pre-imaging before tumor treatment may thus be carried out by:

-   -   (a) administering to the animal or patient a diagnostically        effective amount of a pharmaceutical composition comprising at        least a first detectably-labeled aminophospholipid binding        construct that comprises a diagnostic agent operatively attached        to an antibody, binding protein or ligand, or aminophospholipid        binding fragment thereof, that binds to an aminophospholipid,        preferably phosphatidylserine or phosphatidylethanolamine,        present, expressed, translocated, presented or complexed at the        luminal surface of blood vessels or intratumoral blood vessels        of the vascularized tumor; and    -   (b) subsequently detecting the detectably-labeled        aminophospholipid binding construct bound to an        aminophospholipid, preferably phosphatidylserine or        phosphatidylethanolamine, on the luminal surface of tumor or        intratumoral blood vessels, thereby obtaining an image of the        tumor vasculature.

Cancer treatment may also be carried out by:

-   -   (a) forming an image of a vascularized tumor by administering to        an animal or patient having a vascularized tumor a        diagnostically minimal amount of at least a first        detectably-labeled aminophospholipid binding construct        comprising a diagnostic agent operatively attached to an        antibody, binding protein or ligand, or aminophospholipid        binding fragment thereof, that binds to an aminophospholipid,        preferably phosphatidylserine or phosphatidylethanolamine, on        the luminal surface of tumor or intratumoral blood vessels of        the vascularized tumor, thereby forming a detectable image of        the tumor vasculature; and    -   (b) subsequently administering to the same animal or patient a        therapeutically optimized amount of at least a first therapeutic        agent-targeting agent construct that binds to an        aminophospholipid, preferably phosphatidylserine or        phosphatidylethanolamine, on the tumor or intratumoral blood        vessel luminal surface and thereby destroys the tumor        vasculature.

Imaging and treatment formulations or medicaments are thus provided,which generally comprise:

-   -   (a) a first pharmaceutical composition comprising a        diagnostically effective amount of a detectably-labeled        aminophospholipid binding construct that comprises a detectable        agent operatively attached to an antibody, binding protein or        ligand, or aminophospholipid binding fragment thereof, that        binds to an aminophospholipid, preferably phosphatidylserine or        phosphatidylethanolamine, on the luminal surface of tumor or        intratumoral blood vessels of the vascularized tumor; and    -   (b) a second pharmaceutical composition comprising a        therapeutically effective amount of at least one therapeutic        agent-targeting agent construct, preferably one that binds to        phosphatidylserine or phosphatidylethanolamine.

In such methods and medicaments, advantages will be realized wherein thefirst and second pharmaceutical compositions comprise the same targetingagents, e.g., anti-aminophospholipid antibodies, or fragments thereof,from the same antibody preparation, or preferably, from the sameantibody-producing hybridoma. The foregoing medicaments may also furthercomprise one or more anti-cancer agents.

In the vasculature imaging aspects of the invention, it is recognizedthat the administered detectably-labeled aminophospholipid bindingconstruct, or anti-aminophospholipid antibody-detectable agent, mayitself have a therapeutic effect. Whilst this would not be excluded fromthe invention, the amounts of the detectably-labeled constructs to beadministered would generally be chosen as “diagnostically effectiveamounts”, which are typically lower than the amounts required fortherapeutic benefit.

In the imaging embodiments, as with the therapeutics, the targetingagent may be either antibody-based or binding ligand- or bindingprotein-based. Although not previously connected with tumors or tumorvasculature, detectably labeled aminophospholipid binding ligandcompositions are known in the art and can now, in light of thismotivation and the present disclosure, be used in the present invention.The detectably-labeled annexins of U.S. Pat. No. 5,627,036; WO 95/19791;WO 95/27903; WO 95/34315; WO 96/17618; and WO 98/04294; eachincorporated herein by reference; may thus be employed.

In still further embodiments, the animals or patients to be treated bythe present invention are further subjected to surgery or radiotherapy,or are provided with a therapeutically effective amount of at least afirst anti-cancer agent. The “at least a first anti-cancer agent” inthis context means “at least a first anti-cancer agent in addition tothe therapeutic agent-targeting agent construct of the invention. The“at least a first anti-cancer agent” may thus be considered to be “atleast a second anti-cancer agent”, where the therapeutic agent-targetingagent construct is a first anti-cancer agent. However, this is purely amatter of semantics, and the practical meaning will be clear to those ofordinary skill in the art.

The at least a first anti-cancer agent may be administered to the animalor patient substantially simultaneously with the therapeuticagent-targeting agent construct; such as from a single pharmaceuticalcomposition or from two pharmaceutical compositions administered closelytogether.

Alternatively, the at least a first anti-cancer agent may beadministered to the animal or patient at a time sequential to theadministration of the at least a first therapeutic agent-targeting agentconstruct. “At a time sequential”, as used herein, means “staggered”,such that the at least a first anti-cancer agent is administered to theanimal or patient at a time distinct to the administration of the atleast a first therapeutic agent-targeting agent construct. Generally,the two agents are administered at times effectively spaced apart toallow the two agents to exert their respective therapeutic effects,i.e., they are administered at “biologically effective time intervals”.

The at least a first anti-cancer agent may be administered to the animalor patient at a biologically effective time prior to the therapeuticagent-targeting agent construct, or at a biologically effective timesubsequent to the therapeutic agent-targeting agent construct.Administration of a non-aminophospholipid targeted anti-cancer agent ata therapeutically effective time subsequent to the therapeuticagent-targeting agent construct may be particularly desired wherein theanti-cancer agent is an anti-tumor cell immunotoxin designed to killtumor cells at the outermost rim of the tumor, and/or wherein theanti-cancer agent is an anti-angiogenic agent designed to preventmicrometastasis of any remaining tumor cells. Such considerations willbe known to those of skill in the art.

Administration of one or more non-aminophospholipid targeted anti-canceragents at a therapeutically effective time prior to a therapeuticagent-targeting agent construct may be particularly employed where theanti-cancer agent is designed to increase aminophospholipid expression.This may be achieved by using anti-cancer agents that injure, or induceapoptosis in, the tumor endothelium. Exemplary anti-cancer agentinclude, e.g., taxol, vincristine, vinblastine, neomycin,combretastatin(s), podophyllotoxin(s), TNF-α, angiostatin, endostatin,vasculostatin, α_(v)β₃ antagonists, calcium ionophores, calcium-fluxinducing agents, any derivative or prodrug thereof.

The one or more additional anti-cancer agents may be chemotherapeuticagents, radiotherapeutic agents, cytokines, anti-angiogenic agents,apoptosis-inducing agents or anti-cancer immunotoxins or coaguligands.“Chemotherapeutic agents”, as used herein, refer to classicalchemotherapeutic agents or drugs used in the treatment of malignancies.This term is used for simplicity notwithstanding the fact that othercompounds may be technically described as chemotherapeutic agents inthat they exert an anti-cancer effect. However, “chemotherapeutic” hascome to have a distinct meaning in the art and is being used accordingto this standard meaning.

A number of exemplary chemotherapeutic agents are described herein.Those of ordinary skill in the art will readily understand the uses andappropriate doses of chemotherapeutic agents, although the doses maywell be reduced when used in combination with the present invention. Anew class of drugs that may also be termed “chemotherapeutic agents” areagents that induce apoptosis. Any one or more of such drugs, includinggenes, vectors and antisense constructs, as appropriate, may also beused in conjunction with the present invention.

Anti-cancer immunotoxins or coaguligands are further appropriateanti-cancer agents. “Anti-cancer immunotoxins or coaguligands”, ortargeting-agent/therapeutic agent constructs, are based upon targetingagents, including antibodies or antigen binding fragments thereof, thatbind to a targetable component of a tumor cell, tumor vasculature ortumor stroma, and that are operatively attached to a therapeutic agent,generally a cytotoxic agent (immunotoxin) or coagulation factor(coaguligand). A “targetable component” of a tumor cell, tumorvasculature or tumor stroma, is preferably a surface-expressed,surface-accessible or surface-localized component, although componentsreleased from necrotic or otherwise damaged tumor cells or vascularendothelial cells may also be targeted, including cytosolic and/ornuclear tumor cell antigens.

Both antibody and non-antibody targeting agents may be used, includinggrowth factors, such as VEGF and FGF; peptides containing the tripeptideR-G-D, that bind specifically to the tumor vasculature; and othertargeting components such as annexins and related ligands.

Anti-tumor cell immunotoxins or coaguligands may comprise antibodiesexemplified by the group consisting of B3 (ATCC HB 10573), 260F9 (ATCCHB 8488), D612 (ATCC HB 9796) and KS1/4, said KS1/4 antibody obtainedfrom a cell comprising the vector pGKC2310 (NRRL B-18356) or the vectorpG2A52 (NRRL B-18357).

Anti-tumor stroma immunotoxins or coaguligands will generally compriseantibodies that bind to a connective tissue component, a basementmembrane component or an activated platelet component; as exemplified bybinding to fibrin, RIBS or LIBS.

Anti-tumor vasculature immunotoxins or coaguligands may compriseligands, antibodies, or fragments thereof, that bind to asurface-expressed, surface-accessible or surface-localized component ofthe blood transporting vessels, preferably the intratumoral bloodvessels, of a vascularized tumor. Such antibodies include those thatbind to surface-expressed components of intratumoral blood vessels of avascularized tumor, including aminophospholipids themselves, andintratumoral vasculature cell surface receptors, such as endoglin (TEC-4and TEC-11 antibodies), a TGFβ receptor, E-selectin, P-selectin, VCAM-1,ICAM-1, PSMA, a VEGF/VPF receptor, an FGF receptor, a TIE, α_(v)β₃integrin, pleiotropin, endosialin and MHC Class II proteins. Theantibodies may also bind to cytokine-inducible or coagulant-induciblecomponents of intratumoral blood vessels.

Other anti-tumor vasculature immunotoxins or coaguligands may compriseantibodies, or fragments thereof, that bind to a ligand or growth factorthat binds to an intratumoral vasculature cell surface receptor. Suchantibodies include those that bind to VEGF/VPF (GV39 and GV97antibodies), FGF, TGFβ, a ligand that binds to a TIE, a tumor-associatedfibronectin isoform, scatter factor/hepatocyte growth factor (HGF),platelet factor 4 (PF4), PDGF and TIMP. The antibodies, or fragmentsthereof, may also bind to a ligand:receptor complex or a growthfactor:receptor complex, but not to the ligand or growth factor, or tothe receptor, when the ligand or growth factor or the receptor is not inthe ligand:receptor or growth factor:receptor complex.

Anti-tumor cell, anti-tumor stroma or anti-tumor vasculatureantibody-therapeutic agent constructs may comprise cytotoxic agents suchas plant-, fungus- or bacteria-derived toxins (immunotoxins). Ricin Achain and deglycosylated ricin A chain will often be preferred, andgelonin and angiopoietins are also contemplated. Anti-tumor cell,anti-tumor stroma or anti-tumor vasculature antibody-therapeutic agentconstructs may comprise coagulation factors or second antibody bindingregions that bind to coagulation factors (coaguligands). The operativeassociation with Tissue Factor or Tissue Factor derivatives, such astruncated Tissue Factor, will often be preferred.

The invention still further provides a series of novel therapeuticbinding ligands, binding ligand compositions and pharmaceuticalcompositions, each of which comprise at least a first targeting agentthat binds to an aminophospholipid, operatively attached to at least afirst therapeutic agent, such as a cytotoxin, anti-angiogenic agent orcoagulant. Radiolabels are generally excluded from the binding ligandsand binding ligand compositions; although not from the diagnosticmethods, or even from the therapeutic methods described above.

The targeting agents of the binding ligands preferably bind tophosphatidylethanolamine and/or phosphatidylserine. The entire range ofbinding ligands described above in the context of the therapeutic andcombined methods may be employed in the present compositions. Annexinconjugates and constructs; anti-PS, anti-PE, human, humanized andmonoclonal antibody conjugates and constructs; ricin conjugates; andTissue Factor conjugates and constructs are currently preferred.Compositions comprising one or more anti-PS antibodies operativelyattached to one or more Tissue Factor derivatives, preferably, truncatedTissue Factor, are currently particularly preferred.

Direct or indirect attachment and linkages may be employed in thebinding ligand compositions, including all variations of bispecificantibodies. Operative combinations of a first antigen-binding region ofan antibody that binds to an aminophospholipid, with a secondantigen-binding region of an antibody that binds Tissue Factor or aTissue Factor derivative are also preferred. In the aminophospholipidbinding protein constructs or conjugates, annexins are preferred, withAnnexin V being more preferred, and Annexin V operatively attached totruncated Tissue Factor currently being most preferred.

Components of the invention therefore include an antibody construct,comprising at least a first anti-aminophospholipid antibody, orantigen-binding fragment thereof, operatively attached to at least afirst therapeutic agent; and a bispecific antibody, comprising a firstantigen-binding region that binds to an aminophospholipid operativelyattached to a second antigen-binding region that binds to a therapeuticagent.

The compositions and pharmaceutical compositions may comprise at least afirst and second binding ligand that each comprise at least a firsttargeting agent operatively attached to at least a first therapeuticagent; wherein each targeting agent binds to an aminophospholipid.Compositions and pharmaceutical compositions that comprise at least afirst binding ligand that binds to phosphatidylethanolamine and at leasta second binding ligand that binds to phosphatidylserine are exemplarycombined compositions.

The present invention yet further provides a series of novel therapeutickits, medicaments and/or cocktails for use in conjunction with themethods of the invention. The kits, medicaments and/or cocktailsgenerally comprise a combined effective amount of an anti-cancer agentand a therapeutic agent-targeting agent construct, preferably one thatbinds to phosphatidylserine or phosphatidylethanolamine. Imagingcomponents may also be included.

The kits and medicaments will comprise, preferably in suitable containermeans, a biologically effective amount of at least a first therapeuticagent-targeting agent construct, preferably binding tophosphatidylserine or phosphatidylethanolamine; in combination with abiologically effective amount of at least a first anti-cancer agent. Thecomponents of the kits and medicaments may be comprised within a singlecontainer or container means, or comprised within distinct containers orcontainer means. The cocktails will generally be admixed together forcombined use.

The entire range of therapeutic agent-targeting agent construct, asdescribed above, may be employed in the kits, medicaments and/orcocktails, with annexin conjugates and constructs; anti-PS, anti-PE,human, humanized and monoclonal antibody conjugates and constructs;ricin conjugates; and Tissue Factor conjugates and constructs beingpreferred. The anti-cancer agents are also those as described above,including chemotherapeutic agents, radiotherapeutic agents,anti-angiogenic agents, apoptopic agents, immunotoxins and coaguligands.Agents formulated for intravenous administration will often bepreferred.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A and FIG. 1B. Activity of cell-bound anti-VCAM-1∘tTF in vitro.FIG. 1A. Binding of anti-VCAM-1∘tTF coaguligand to unstimulated(control) and IL-1α-activated bEnd.3 cells. FIG. 1B. Generation offactor Xa by cell-bound anti-VCAM-1∘tTF coaguligand.

FIG. 2. Retardation of growth of L540 tumors in mice treated withanti-VCAM-1∘tTF. L540 tumor bearing mice were injected i.v. with eithersaline, 20 μg of anti-VCAM-1∘tTF, 4 μg of unconjugated tTF or 20 μg ofcontrol IgG∘tTF. Injections were repeated on day 4 and 8 after the firsttreatment. Tumors were measured daily. Mean tumor volume and SD of 8mice per group is shown.

FIG. 3. Annexin V blocks coaguligand activation of Factor X in vitro.IL-1α-stimulated bEnd.3 cells were incubated with anti-VCAM-∘tTFcoaguligand in 96-well microtiter plates, as described in Example V.Annexin V was added at concentrations ranging from 0.1 to 10 μg/ml (asshown) and cells were incubated for 30 min. before addition of dilutedProplex T. The amount of Factor Xa generated in the presence or absenceof Annexin V was determined using a chromogenic substrate, as describedin Example V.

FIG. 4A and FIG. 4B. Anti-tumor effects of naked anti-PS antibodies inanimals with syngeneic and xenogeneic tumors. 1×10⁷ cells of murinecolorectal carcinoma Colo 26 (FIG. 4A) or human Hodgkin's lymphoma L540(FIG. 4B) were injected subcutaneously into the right flank of Balb/cmice (FIG. 4A) or male CB17 SCID mice (FIG. 4B), respectively. Tumorswere allowed to grow to a size of about 0.6–0.9 cm³ and then the mice (4animals per group) were injected i.p. with 20 μg of naked anti-PSantibody (open squares) or saline (open circles) (control mouse IgM gavesimilar results to saline.). Treatment was repeated 3 times with a 48hour interval. Animals were monitored daily for tumor measurements andbody weight. Tumor volume was calculated as described in Example VII.Mice were sacrificed when tumors had reached 2 cm³, or earlier if tumorsshowed signs of necrosis or ulceration.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

A. Tumor Destruction Using VCAM-1 Coaguligand

Solid tumors and carcinomas account for more than 90% of all cancers inman. Although the use of monoclonal antibodies and immunotoxins has beeninvestigated in the therapy of lymphomas and leukemias (Vitetta et al.,1991), these agents have been disappointingly ineffective in clinicaltrials against carcinomas and other solid tumors (Abrams and Oldham,1985). A principal reason for the ineffectiveness of antibody-basedtreatments is that macromolecules are not readily transported into solidtumors. Even once within a tumor mass, these molecules fail todistribute evenly due to the presence of tight junctions between tumorcells, fibrous stroma, interstitial pressure gradients and binding sitebarriers (Dvorak et al., 1991).

In developing new strategies for treating solid tumors, the methods thatinvolve targeting the vasculature of the tumor, rather than the tumorcells, offer distinct advantages. An effective destruction or blockadeof the tumor vessels arrests blood flow through the tumor and results inan avalanche of tumor cell death. Antibody-toxin and antibody-coagulantconstructs have already been effectively used in the specific targetingand destruction of tumor vessels, resulting in tumor necrosis (Burrowset al., 1992; Burrows and Thorpe, 1993; WO 93/17715; WO 96/01653; U.S.Pat. Nos. 5,855,866; 5,877,289; 5,965,132; 6,004,555; and 6,093,399;

Where antibodies, growth factors or other binding ligands are used tospecifically deliver a coagulant to the tumor vasculature, such agentsare termed “coaguligands”. A currently preferred coagulant for use incoaguligands is truncated Tissue Factor (tTF) (Huang et al., 1997; WO96/01653; U.S. Pat. No. 5,877,289). TF is the major initiator of bloodcoagulation (Ruf et al., 1991). At sites of injury, Factor VII/VIIa inthe blood comes into contact with, and binds to, TF on cells in theperivascular tissues. The TF:VIIa complex, in the presence of thephospholipid surface, activates factors IX and X. This, in turn, leadsto the formation of thrombin and fibrin and, ultimately, a blood clot(Ruf and Edgington, 1994).

The recombinant, truncated form of tissue factor (tTF), lacking thecytosolic and transmembrane domains, is a soluble protein that has aboutfive orders of magnitude lower coagulation inducing ability than nativeTF (Stone et al., 1995; Huang et al., 1997). This is because TF needs tobe associated with phospholipids for the complex with VIIa to activateIXa or Xa efficiently. However, when tTF is delivered to tumor vascularendothelium by means of a targeting antibody or agent, it is broughtback into proximity to a lipid surface and regains thrombogenic activity(Huang et al., 1997; U.S. Pat. Nos. 5,877,289; 6,004,555; and 6,093,399)coaguligand is thus created that selectively thromboses tumorvasculature.

Truncated TF has several advantages that commend its use in vasculartargeted coaguligands: human tTF is readily available, and the humanprotein will have negligible or low immunogenicity in man; human tTF isfully functional in experimental animals, including mice; and targetedtTF is highly potent because it triggers the activation of a cascade ofcoagulation proteins, giving a greatly amplified effect (U.S. Pat. Nos.5,877,289; 6,004,555 and 6,093,399)

A range of suitable target molecules that are available on tumorendothelium, but largely absent from normal endothelium, have beendescribed. For example, expressed targets may be utilized, such asendoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a TIE, a ligandreactive with LAM-1, a VEGF/VPF receptor, an FGF receptor, α_(v)β₃integrin, pleiotropin or endosialin (U.S. Pat. Nos. 5,855,866;5,5,877,289 and 6,004,555; Burrows et al., 1992; Burrows and Thorpe,1993; Huang et al., 1997; Liu et al., 1997; Ohizumi et al., 1997; eachincorporated herein by reference).

Adsorbed targets are another suitable group, such as VEGF, FGF, TGFβ,HGF, PF4, PDGF, TIMP, a ligand that bind to a TIE or a tumor-associatedfibronectin isoform (U.S. Pat. Nos. 5,877,289; 5,965,132 and 6,004,555;each incorporated herein by reference). Fibronectin isoforms are ligandsthat bind to the integrin family of receptors. Tumor-associatedfibronectin isoforms are targetable components of both tumor vasculatureand tumor stroma. The monoclonal antibody BC-1 (Carnemolla et al, 1989)specifically binds to tumor-associated fibronectin isoforms.

Other targets inducible by the natural tumor environment or followingintervention by man are also targetable entities, as described in U.S.Pat. Nos. 5,776,427, 5,863,538 and 6,036,955; paid; each incorporatedherein by reference). When used in conjunction with prior suppression innormal tissues and tumor vascular induction, MHC Class II antigens mayalso be employed as targets (U.S. Pat. Nos. 5,776,427; 5,863,538;6,004,554 and 6,036,955; each incorporated herein by reference).

One currently preferred target for clinical applications is vascularendothelial adhesion molecule-1 (VCAM-1) (U.S. Pat. Nos. 5,855,866,5,877,289, 6,004,555 and 6,093,399; each incorporated herein byreference). VCAM-1 is a cell adhesion molecule that is induced byinflammatory cytokines IL-α, IL-4 (Thornhill et al., 1990) and TNFα(Munro, 1993) and whose role in vivo is to recruit leukocytes to sitesof acute inflammation (Bevilacqua, 1993).

VCAM-1 is present on vascular endothelial cells in a number of humanmalignant tumors including neuroblastoma (Patey et al., 1996), renalcarcinoma (Droz et al., 1994), non-small lung carcinoma (Staal-van denBrekel et al., 1996), Hodgkin's disease (Patey et al., 1996), andangiosarcoma (Kuzu et al., 1993), as well as in benign tumors, such asangioma (Patey et al., 1996) and hemangioma (Kuzu et al., 1993).Constitutive expression of VCAM-1 in man is confined to a few vessels inthe thyroid, thymus and kidney (Kuzu et al., 1993; Bruijn and Dinklo,1993), and in the mouse to vessels in the heart and lung (Fries et al.,1993).

Certain of the data presented herein even further supplement thoseprovided in U.S. Pat. Nos. 5,855,866, 5,877,289 and 6,004,555; eachincorporated herein by reference) and show the selective induction ofthrombosis and tumor infarction resulting from administration of ananti-VCAM-1•tTF coaguligand. The results presented were generated usingmice bearing L540 human Hodgkin lymphoma. When grown as a xenograft inSCID mice, this tumor shows close similarity to the human disease withrespect to expression of inflammatory cytokines (Diehl et al., 1985) andthe presence of VCAM-1 and other endothelial cell activation moleculeson its vasculature.

Using a covalently-linked anti-VCAM-1∘tTF coaguligand, in which tTF wasdirectly linked to the anti-VCAM-1 antibody, it is shown herein that thecoaguligand localizes selectively to tumor vessels, induces thrombosisof those vessels, causes necrosis to develop throughout the tumor andretards tumor growth in mice bearing solid L540 Hodgkin tumors. Tumorsgenerally needed to be at least about 0.3 cm in diameter to respond tothe coaguligand, because VCAM-1 was absent from smaller tumors.Presumably, in small tumors, the levels of cytokines secreted by tumorcells or host cells that infiltrate the tumor are too low for VCAM-1induction. This is in accordance with the studies in U.S. Pat. Nos.5,855,866, 5,877,289, 5,776,427, 6,004,555 and 6,036,955, where theinventions were shown to be most useful in larger solid tumors.

Although VCAM-1 staining was initially observed more in the periphery ofthe tumor, the coaguligand evidently bound to and occluded bloodtransporting vessels—as it was capable of curtailing blood flow in alltumor regions. Furthermore, one of the inventors contemplates that thethrombin generation caused by the initial administration of thecoaguligand likely leads to further VCAM-1 induction on central vessels(Sluiter et al., 1993), resulting in an amplified signal and evidentdestruction of the intratumoral region. This type of coagulant-inducedexpression of further targetable markers, and hence signalamplification, is also disclosed in U.S. Pat. No. 6,036,955.

B. Mechanism of VCAM-1-Targeted Tumor Destruction

As shown herein, although localization to VCAM-1-expressing vessels inthe heart and lungs of mice was observed upon administration of ananti-VCAM-1 coaguligand, this construct did not induce thrombosis insuch non-tumor sites. Furthermore, the anti-VCAM-1 coaguligand was nomore toxic to mice than was a control coaguligand of irrelevantspecificity, again indicating that the constitutive expression of VCAM-1on heart and lung vessels did not lead to toxicity. This data isimportant to the immediate clinical progress of coaguligand therapy,given that VCAM-1 is a naturally occurring marker of tumor vascularendothelium in humans. However, this phenomenon also provided theinventors with a unique insight, leading to other approaches for tumorvasculature destruction.

The inventors sought to understand the mechanism behind the ability ofthe anti-VCAM-1 coaguligand to bind to the VCAM-1 constitutivelyexpressed on blood vessels in the heart and lungs, and yet not to causethrombosis in those vessels. There are numerous scientific possibilitiesfor this empiric observation, generally connected with the prothromboticnature of the tumor environment and any fibrinolytic predisposition inthe heart and lungs.

Generally, there is a biological equilibrium between the coagulationsystem (fibrin deposition) and the fibrinolytic system (degradation offibrin by enzymes). However, in malignant disease, particularlycarcinomas, this equilibrium is disrupted, resulting in the abnormalactivation of coagulation (hypercoagulability or the “prothromboticstate”). Evidence also indicates that various components of thesepathways may contribute to the disorderly characteristics of malignancy,such as proliferation, invasion, and metastasis (Zacharski et al.,1993).

Donati (1995) reviewed the complex interplay between the originalclinical observations of thrombotic complications of malignant diseases,and the subsequent progress in the cell biology and biochemistry oftumor cell activities. However, despite extensive research, a clearmolecular explanation for the prothrombotic nature of the tumorenvironment could not be provided (Donati, 1995). Donati did emphasize,though, the role of tumor cells in this process. It was explained thattumor cells express procoagulant activities, such as tissuethromboplastin and cancer procoagulant (CP) (Donati, 1995). WO 91/07187also reported a procoagulant activity of tumor cells.

Numerous other studies have also identified the tumor cells themselvesas being responsible for the prothrombotic state within a tumor. Forexample, Nawroth et al. (1988) reported that factor(s) elaborated bysarcoma cells enhance the procoagulant response of nearby endothelium toTNF. These authors reported that fibrin formation occurred throughoutthe tumor vascular bed 30 minutes after TNF infusion, but that fibrindeposition and platelet aggregates were not observed in normalvasculature (Nawroth et al., 1988). TNF was later shown to enhance theexpression of tissue factor on the surface of endothelial cells (Murrayet al., 1991). This was proposed to explain earlier studies showing thatcultured endothelial cells incubated with recombinant TNF have enhancedprocoagulant activity, tissue factor, and concomitant suppression of theprotein C pathway, an anti-thrombotic mechanism that functions on thesurface of quiescent endothelial cells (Nawroth et al., 1985; Nawrothand Stern, 1986).

Data from Sugimura et al. (1994) also implicated tumor cells as the keycomponents of the procoagulant activity of the tumor. It was reportedthat four tumor cell lines were able to support different stages of theextrinsic pathway of coagulation (Sugimura et al., 1994). Another studyreported that a human ovarian carcinoma cell line, OC-2008,constitutively expressed surface membrane Tissue Factor activity andexhibited cell surface-dependent prothrombinase complex activity (Rao etal., 1992). Connor et al. (1989) further suggested that it is thepathologic cells that control coagulation. Their results indicated thattumorigenic, undifferentiated murine erythroleukemic cells exhibit a 7-to 8-fold increase in the potency of their procoagulant activity (Connoret al., 1989).

Zacharski et al. (1993) also focused on tumor cells and sought to definethe mode of interaction of ovarian carcinoma cells with the coagulation(procoagulant-initiated) and fibrinolysis (urokinase-type plasminogenactivator-initiated, u-PA) pathways. They reported that tumor cellsexpressed Tissue Factor and coagulation pathway intermediates thatresulted in local thrombin generation—as evidenced by the conversion offibrinogen, present in tumor connective tissue, to fibrin that was foundto hug the surfaces of tumor nodules and individual tumor cells.Detected fibrin could not be accounted for on the basis of necrosis or alocal inflammatory cell infiltrate (Zacharski et al., 1993). Theseauthors concluded that there exists a dominant tumor cell-associatedprocoagulant pathway that leads to thrombin generation andhypercoagulability.

Other hypotheses have proposed that it is changes in the tumor bloodvessels that render these vessels better able to support the formationof thrombi and/or less able to dissolve fibrin. For example, tumorvessels have been reported to exhibit upregulation of Tissue Factor,down-regulation of plasminogen activators and/or upregulation of theinhibitor of plasminogen activators, PAI-1 (Nawroth and Stern, 1986;Nawroth et al, 1988). Such effects are believed to be magnified by tumorderived factors (Murray et al., 1991; Ogawa et al., 1990), possiblyVEGF.

For example, Ogawa et al. (1990) reported that hypoxia causedendothelial cell surface coagulant properties to be shifted to promoteactivation of coagulation. This was accompanied by suppression of theanticoagulant cofactor, thrombomodulin, and induction of an activator offactor X, distinct from the classical extrinsic and intrinsic systems(Ogawa et al., 1990). Also, there could be an increase in the localconcentration of Factors VIIa, IXa, Xa, or other molecules that interactwith TF, within the tumor vessels, thus encouraging thrombosis.

Additionally, platelets are a major component of any procoagulant state.Recently, the procoagulant potential of platelets has been linked totheir ability to shed procoagulant microparticles from the plasmamembrane (Zwaal et al., 1989; 1992; Dachary-Prigent et al, 1996). It hasbeen proposed that an increased proportion of circulatingmicroparticles, vesicles or membrane fragments from plateletscontributes to ‘prethrombotic’ (prothrombotic) states in variouspathological conditions (Zwaal et al., 1989; 1992; Dachary-Prigent etal., 1996, pp. 159 and references cited therein). McNeil et al (1990)also reported that β₂-GPI exerts multiple inhibitory effects oncoagulation and platelet aggregation. Tumor platelet biology could thusexplain the effectiveness of the anti-VCAM-1 coaguligand.

Further tenable explanations include the simple possibility that VCAM-1is expressed at higher levels in tumor vessels than on blood vessels inthe heart and lungs, probably due to induction by tumor-derivedcytokines, and that binding to the healthy vessels cannot tip thebalance into sustained thrombosis. Also the fibrinolytic mechanismscould be upregulated in the heart, as exemplified by increased TissueFactor pathway inhibitor (TFPI), increased plasminogen activators,and/or decreased plasminogen activator inhibitors. Should thefibrinolytic physiology of the heart and lung vessels prove to be themajor reason underlying the tumor-specific effects of the anti-VCAM-1coaguligand, this would generally preclude the development of additionalanti-tumor therapies targeted to unique aspects of tumor biology.

Despite all the possible options, the inventors reasoned that thefailure of the anti-VCAM-1 coaguligand to cause thrombosis in vessels ofnormal tissues was due to the absence of the aminophospholipid,phosphatidylserine (PS), from the luminal surface of such vessels. Tocomplete the theory, therefore, not only would phosphatidylserine haveto be shown to be absent from these normal vessels, but its presence onthe luminal side of tumor-associated vessels would have to beconclusively demonstrated.

The inventors therefore used immunohistochemical staining to evaluatethe distribution of a monoclonal anti-phosphatidylserine (anti-PS)antibody injected intravenously into tumor-bearing mice. These studiesrevealed that the VCAM-1 expressing vessels in the heart and lungslacked PS, whereas the VCAM-1 expressing vessels in the tumor expressedPS. The need for surface PS expression in coaguligand action is furtherindicated by the inventors' finding that annexin V, which binds to PS,blocks anti-VCAM-1∘tTF coaguligand action, both in vitro and in vivo.

The lack of thrombotic effect of the anti-VCAM-1 coaguligand on normalheart and lung vessels can thus be explained, at least in part: theabsence of the aminophospholipid, phosphatidylserine, means that thenormal vessels lack a procoagulant surface upon which coagulationcomplexes can assemble. In the absence of surface PS, anti-VCAM-1∘tTFbinds to VCAM-1 expressing heart and lung vessels, but cannot inducethrombosis. In contrast, VCAM-1 expressing vessels in the tumor showcoincident expression of surface PS. The coaguligand thus binds to tumorvessels and activates coagulation factors locally to form an occlusivethrombus.

In addition to delineating the tumor-specific thrombotic effects ofanti-VCAM-1 coaguligands, the specific expression of theaminophospholipid, phosphatidylserine, on the luminal surface of tumorblood vessels also allowed the inventors to explain the prothromboticphenotype observed, but not understood, in earlier studies (Zacharski etal., 1993; Donati, 1995). Rather than being predominantly due to tumorcells or elaborated factors; platelets, procoagulant microparticles ormembrane fragments; or due to imbalances in thromboplastin,thrombomodulin, cancer procoagulant, Tissue Factor, protein C pathway,plasminogen activators or plasminogen activator inhibitors (e.g.,PAI-1), the inventors' studies indicate that it is PS expression thatplays a significant role in the prothrombotic state of tumorvasculature.

C. Aminophospholipids as Markers of Tumor Vasculature

Following their discovery that the representative aminophospholipid,phosphatidylserine, was specifically expressed on the luminal surface oftumor blood vessels, but not in normal blood vessels, the inventorsreasoned that aminophospholipids had potential as targets fortherapeutic intervention. The present invention therefore providescompositions and methods for the targeted delivery of therapeutic agentsto aminophospholipid membrane constituents, particularlyphosphatidylserine (PS) and phosphatidylethanolamine (PE). Althoughanti-tumor effects from aminophospholipid-targeted delivery aredemonstrated herein, using art-accepted animal models, the ability ofaminophospholipids to act as safe and effective targetable markers oftumor vasculature could not have been predicted from previous studies.

For example, although tumor vessels are generally prothrombotic innature, as opposed to other blood vessels, it is an inherent property ofthe tumor to maintain a network of blood vessels in order to deliveroxygen and nutrients to the tumor cells. Evidently, tumor-associatedblood vessels cannot be so predisposed towards thrombosis that theyspontaneously and readily support coagulation, as such coagulation wouldnecessarily cause the tumor to self-destruct. It is thus unexpected thatany thrombosis-associated tumor vessel marker, such as the presentlyidentified phosphatidylserine, could be discovered that is expressed inquantities sufficient to allow effective therapeutic intervention bytargeting, and yet is expressed at levels low enough to ordinarilymaintain blood flow through the tumor.

The present identification of aminophospholipids as safe and effectivetumor vasculature targets is even more surprising given (1) the previousspeculations regarding the role of other cell types and/or variousfactors, activators and inhibitors underlying the complex, prothromboticstate of the tumor (as discussed above); and (2) the confusing andcontradictory state of the art concerning aminophospholipid biology, interms of both expression and function in various cell types.

Phosphatidylserine and phosphatidylethanolamine are normally segregatedto the inner surface of the plasma membrane bilayer in different cells(Gaffet et al., 1995; Julien et al., 1995). In contrast, the outerleaflet of the bilayer membrane is rich in phosphatidylcholine analogs(Zwaal et al., 1989; Gaffet et al., 1995). This lipid segregationcreates an asymmetric transbilayer. Although the existence of membraneasymmetry has been discussed for some time, the reason for its existenceand the mechanisms for its generation and control are poorly understood(Williamson and Schlegel, 1994), particularly in cells other thanplatelets.

There are even numerous conflicting reports regarding the presence orabsence of PS and PE in different cells and tissues, let aloneconcerning the likely role that these aminophospholipids may play. Forexample, the many PS studies conducted with platelets, key components inblood coagulation (Dachary-Prigent et al., 1996), have yielded highlyvariable results. Bevers et al. (1982) measured the plateletprothrombin-converting activity of non-activated platelets aftertreatment with various phospholipases or proteolytic enzymes. Theyconcluded that negatively charged phosphatidylserine, and possiblyphosphatidylinositol, were involved in the prothrombin-convertingactivity of non-activated platelets (Bevers et al., 1982).

Bevers et al. (1983) then reported an increased exposure ofphosphatidylserine, and a decreased exposure of sphingomyelinase, inactivated platelets. However, these alterations were much less apparentin platelets activated either by thrombin or by collagen alone, incontrast to collagen plus thrombin, diamide, or a calcium ionophore(Bevers et al., 1983). The surface expression of PS in response todiamide was contradicted by studies in erythrocytes, which showed nodiamide-stimulated PS exposure (de Jong et al., 1997). While echoingtheir earlier results, Bevers and colleagues then later reported thatchanges in the plasma membrane-cytoskeleton interaction, particularlyincreased degradation of cytoskeletal actin-binding protein, wasimportant to platelet surface changes (Bevers et al., 1985; pages368–369).

Maneta-Peyret et al. (1989) also reported the detection of PS on humanplatelets. These authors noted that the platelet procoagulant surfacecould be formed by negatively charged phospholipids, such asphosphatidylserine and phosphatidylethanolamine (generally neutral orzwitterionic), or both. The role of phosphatidylserine in the process ofcoagulation has been questioned in favor of phosphatidylethanolamine(Maneta-Peyret et al., 1989; Schick et al., 1976; 1978). For example,studies have reported that 18% of phosphatidylethanolamine becomessurface-accessible after 2 hours, in contrast to zero phosphatidylserine(Schick et al., 1976).

Ongoing studies with platelets were also reported as showing a further16% increase in phosphatidylethanolamine exposure after thrombintreatment, with no increase in the phosphatidylserine levels (Schick etal., 1976). Therefore, PS was said not to be a component of thefunctional surface of the platelet plasma membrane (Schick et al., 1976;1978). Nonetheless, current evidence does seem to indicate that both PSand PE are involved in the phospholipid asymmetry observed in the outermembrane of platelets and erythrocytes, and that PS is involved in theprocoagulant activity of platelets (Gaffet et al., 1995; de Jong et al.,1997; U.S. Pat. No. No. 5,627,036).

The mechanisms for achieving and maintaining differentialaminophospholipid distribution, let alone the functional significance ofdoing so, have long been the subject of controversial speculations. Inreviewing the regulation of transbilayer phospholipid movement,Williamson and Schlegel (1994) indicated that elevating intracellularCa²⁺ allows the major classes of phospholipids to move freely across thebilayer, scrambling lipids and dissipating asymmetry. de Jong et al.(1997) also reported that an increase of intracellular calcium leads toa rapid scrambling of the lipid bilayer and the exposure of PS, whichcould be partially inhibited by cellular oxidation. The interaction ofaminophospholipids with cytoskeletal proteins has also been proposed asa mechanism for regulating membrane phospholipid asymmetry (Zwaal etal., 1989).

Gaffet et al. (1995) stated that the transverse redistribution ofphospholipids during human platelet activation is achieved by avectorial outflux of aminophospholipids, not counterbalanced by a rapidreciprocal influx of choline head phospholipids, i.e. not scrambling.They suggested that the specific vectorial outflux of aminophospholipidscould be catalyzed by a “reverse aminophospholipid translocase” activity(Gaffet et al., 1995). An alternative hypothesis would be that theactivity of an inward translocase was inhibited. Zwaal et al. (1989)proposed the involvement of a phospholipid-translocase that catalyzedboth the outward and inward movement of aminophospholipids.

The presence of an energy- and protein-dependent aminophospholipidtranslocase activity that transports phosphatidylethanolamine from theouter to the inner leaflet of the lipid bilayer was reported by Julienet al. (1993). They then showed that the aminophospholipid translocaseactivity could also transfer phosphatidylserine, and that the activitycould be maintained, suppressed and restored depending on the conditionsof cell incubation (Julien et al., 1993), and inhibited by the tumorpromoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Julien et al.,1997).

A 35 kD phospholipid scramblase that promotes the Ca²⁺-dependentbidirectional movement of phosphatidylserine and other phospholipids wasrecently cloned from a cDNA library (Zhou et al., 1997). This “PLscramblase” protein is a proline-rich, type II plasma membrane proteinwith a single transmembrane segment near the C terminus. Subsequentstudies confirmed that this protein was responsible for the rapidmovement of phospholipids from the inner to the outer plasma membraneleaflets in cells exposed to elevated cytosolic calcium concentrations(Zhao et al., 1998).

The aminophospholipid translocase activity reported by Julien et al.(1993; 1997), which transports PS and PE from the outer to the innerleaflet, is different to the bidirectional Ca²⁺-dependent scramblase(Zhou et al., 1997; Zhao et al., 1998). The scramblase is activated byCa²⁺, and mostly functions to move PS from the inner to the outerleaflet in response to increased Ca²⁺ levels. It is now generallybelieved that the aminophospholipid translocase maintains membraneasymmetry during normal conditions, but that the scramblase is activatedby Ca²⁺ influx, over-riding the translocase and randomizingaminophospholipid distribution.

The normal segregation of PS and PE to the inner surface of the plasmamembrane is thus now generally accepted, and certain membrane componentsinvolved in the asymmetric processes have even been identified. However,doubts remain about the conditions, mechanisms and cell types that arecapable of re-locating aminophospholipids to the outer leaflet of themembrane, and the biological implications of such events.

Contradictory reports concerning aminophospholipid expression are notlimited to studies of platelets. Phosphatidylserine andphosphatidylethanolamine are generally about 7% and about 10%,respectively, of the phospholipid composition of cultured humanendothelial cells from human artery, saphenous and umbilical vein (7.1%and 10.2%, respectively; Murphy et al., 1992). However, an importantexample of the contradictions in the literature concerns the ability ofanti-PS antibodies to bind to endothelial cells (Lin et al., 1995).

The anti-PS antibodies present in recurrent pregnancy loss (Rote et al.,1995; Rote, 1996; Vogt et al., 1996; Vogt et al., 1997) were believed tomodulate endothelial cell function, without evidence of binding toendothelial cells. In an attempt to explain this discrepancy, Lin et al.(1995) tried but failed to demonstrate anti-PS antibody binding toresting endothelial cells. They concluded that PS antigenic determinantsare not expressed on the surface of resting endothelial cells, althougha PS-dependent antigenic determinant was associated withcytoskeletal-like components in acetone-fixed cells (Lin et al., 1995).

Van Heerde et al. (1994) reported that vascular endothelial cells invitro can catalyze the formation of thrombin by the expression ofbinding sites at which procoagulant complexes can assemble. In contrastto other studies with activated platelets (Bevers et al., 1982; 1983;1985; Maneta-Peyret et al., 1989; Schick et al., 1976; 1978), stimulatedHUVEC endothelial cells did not exhibit an increase in PS binding sitesas compared to quiescent cells (Van Heerde et al., 1994).Phosphatidylserine was reported to be necessary for Factor Xa formationvia the extrinsic as well as the intrinsic route (Van Heerde et al.,1994). Nonetheless, Brinkman et al. (1994) published contradictoryresults, indicating that other membrane constituents besides negativelycharged phospholipids are involved in endothelial cell mediated,intrinsic activation of factor X.

Ravanat et al. (1992) also studied the catalytic potential ofphospholipids in pro- and anti-coagulant reactions in purified systemsand at the surface of endothelial cells in culture after stimulation.Their seemingly contradictory results were proposed to confirm a rolefor phospholipid-dependent mechanisms in both procoagulant Tissue-Factoractivity and anticoagulant activities (activation of protein C by thethrombin-thrombomodulin complex and by Factor Xa) (Ravanat et al.,1992). The Ravanat et al. (1992) results were also said to provideevidence of phospholipid exposure during activation of human endothelialcells, which was not observed by Van Heerde et al. (1994) or Brinkman etal. (1994). However, they did note that anionic phospholipids are ofrestricted accessibility in the vicinity of cellular Tissue Factor. Thesituation is further complicated as, even after Tissue Factor induction,other events are likely necessary for coagulation, as the Tissue Factorremains inaccessible, being under the cell.

Ravanat et al. (1992) went on to suggest that the different extent ofinhibition of Tissue Factor and thrombomodulin activities on stimulatedendothelial cells means that the cofactor environments differ for theoptimal expression of these opposite cellular activities. However, theacknowledged difficulties in trying to reproduce exact cellularphospholipid environments (Ravanat et al., 1992), raise the possibilityof artifactual data from these in vitro studies. Indeed, irrespective ofthe Ravanat et al. (1992) data, it is generally acknowledged thatmeaningful information regarding tumor biology, and particularlytherapeutic intervention, can only be gleaned from in vivo studies intumor-bearing animals, such as those conducted by the present inventors.

In addition to the disagreements regarding aminophospholipid expression,as discussed above, there are also conflicting reports concerning thefunction of aminophospholipids in various cell types. Although it is nowgenerally accepted that PS expression on activated platelets isconnected with the procoagulant surface, in discussing the physiologicalsignificance of membrane phospholipid asymmetry in platelets and redblood cells, Zwaal et al. (1989) highlighted other important functions.Moreover, Toti et al. (1996) stated that the physiological implicationsof a loss of asymmetric phospholipid distribution remain poorlyunderstood in cell types other than blood cells.

Zwaal et al. (1989) stated that the membrane phospholipid asymmetry ofplatelets and red cells is undone when the cells are activated invarious ways, presumably mediated by the increased transbilayer movementof phospholipids. These changes, coupled with the release of shedmicroparticles, were explained to play a role in local blood clottingreactions. A similar phenomenon was described to occur in sickled redcells: phospholipid vesicles breaking off from reversibly sickled cellscontribute to intravascular clotting in the crisis phase of sickle celldisease (Zwaal et al., 1989).

Both Zwaal et al. (1989) and Williamson and Schlegel (1994) haveindicated that the physiological significance of surface phospholipidchanges is not restricted to hemostasis. In fact, the surface exposureof PS by blood cells was said to significantly alter their recognitionby the reticuloendothelial system, and was to likely represent at leastpart of the homeostatic mechanism for the clearance of blood cells fromthe circulation (Zwaal et al., 1989). Thus, PS acts as a signal for theelimination of activated platelets after bleeding has stopped.Recognition of PS exposed on sickle cells and malarially infected cellsby phagocytes and macrophages explains their counter-pathophysiologicaleffects (Zwaal et al., 1989). Furthermore, PS-dependent phagocytosismarks virally infected cells for phagocytic uptake (WO 97/17084). Thesurface expression of aminophospholipids could also confer “fusioncompetence” to a cell (Williamson and Schlegel, 1994).

Williamson and Schlegel (1994) also speculated that there is a moregeneral raison d'être for lipid asymmetry. For example, although thedifferent head groups have received most attention, it could well bethat fatty acid asymmetry is the important factor (Williamson andSchlegel, 1994). A further hypothesis is that the asymmetricdistribution of transbilayer phospholipids has no function in itself,but that it is the dynamic process of lipid movement that is importantto biological systems (Williamson and Schlegel, 1994).

Many groups have reported that tumor cells are responsible for theprothrombinase activity of the tumor (Connor et al., 1989; Rao et al.,1992; Zacharski et al., 1993; Sugimura et al, 1994; Donati, 1995). Thiscould have been reasoned to be due to PS (WO 91/07187). However, theresults of Sugimura et al. (1994) argue against this: they reported thatalthough both the prothrombinase activity and total procoagulantactivity of the tumorigenic cells, HepG2 and MKN-28, fell on reachingconfluency, the PS levels remained constant.

Rather than supporting a role for tumor cell PS in prothrombinaseactivity, Connor et al. (1989) suggested that the increased expressionof PS in tumorigenic cells is relevant to their ability to be recognizedand bound by macrophages. Utsugi et al. (1991) similarly proposed thatthe presence of PS in the outer membrane of human tumor cells explainstheir recognition by monocytes.

Jamasbi et al. (1994) suggested a totally different role for lipidcomponents in tumorigenic cells, proposing that the lipids interferewith tumor antigen accessibility. Thus, tumor cell lipids would act tomodify the tumor cell surface antigen(s), thus protecting the tumorcells from host immune destruction (Jamasbi et al., 1994). Thishypothesis is not unlike that proposed by Qu et al. (1996), in terms ofendothelial cells. These authors showed that T cells adhered tothrombin-treated human umbilical endothelial cells by virtue of bindingto PS (Qu et al., 1996).

It has thus been proposed that PS-mediated T cell adhesion toendothelial cells in vivo is important to both immune surveillance, andalso to the disease processes of atherosclerosis (Qu et al., 1996;Moldovan et al., 1994). Bombeli et al. (1997) and Flynn et al. (1997)also suggested that cells within atherosclerotic plaques may contributeto disease progression by exposing PS, although this was based solely onin vitro studies. Qu et al. (1996) and Moldovan et al. (1994) evenhinted at an approach opposite to that of the present invention, i.e.,the manipulation of phosphatidylserine interactions as an anticoagulantapproach. U.S. Pat. No. 5,658,877 and No. 5,296,467 have proposedannexin (or “annexine”) for use as anti-endotoxins and anti-coagulants.U.S. Pat. No. 5,632,986 (incorporated herein by reference) suggests theuse of the phosphatidylserine-binding ligand, annexin V, as a conjugatewith a component, such as urokinase, that lyses thrombi.

Toti et al. (1996) suggested that Scott syndrome, an inherited bleedingdisorder, may reflect the deletion or mutation of a putative outwardphosphatidylserine translocase or scramblase”. Although an interestingnotion, Stout et al. (1997) later isolated a membrane protein from Scotterythrocytes that exhibited normal PL scramblase activity whenreconstituted in vesicles with exogenous PLs. It was suggested that thedefect in Scott syndrome is related to an altered interaction of Ca²⁺with PL scramblase on the endofacial surface of the cell membrane, dueeither to an intrinsic constraint upon the protein, preventinginteraction with Ca²⁺ in situ, or due to an unidentified inhibitor orcofactor in the Scott cell that is dissociated by detergent (Stout etal., 1997).

More variable results have been reported in connection with the possiblerole of PS in apoptosis. Williamson and Schlegel (1994) discussed thetheme of PS as a marker of programmed cell death (PCD or apoptosis). Itis generally accepted that programmed cell death, at least in thehematopoietic system, requires the phagocytic sequestration of theapoptopic cells before the loss of membrane integrity or “rupture”. Theloss of membrane asymmetry in apoptopic cells, and particularly theappearance of PS in the external leaflet, was proposed to be the triggerfor their recognition by phagocytic macrophages (Williamson andSchlegel, 1994).

Martin et al. (1995) further reported PS externalization to be an earlyand widespread event during apoptosis of a variety of murine and humancell types, regardless of the initiating stimulus. They also indicatedthat, under conditions in which the morphological features of apoptosiswere prevented (macromolecular synthesis inhibition, overexpression ofBcl-2 or Ab1), the appearance of PS on the external leaflet of theplasma membrane was similarly prevented (Martin et al., 1995).

However, other analyses argue against the Williamson and Schlegel (1994)and Martin et al. (1995) proposals to some extent (Vermes et al., 1995).Although these authors indicate that the translocation of PS to theouter membrane surface is a marker of apoptosis, they reason that thisis not unique to apoptosis, but also occurs during cell necrosis. Thedifference between these two forms of cell death is that during theinitial stages of apoptosis the cell membrane remains intact, while atthe very moment that necrosis occurs the cell membrane loses itsintegrity and becomes leaky. Therefore, according to this reasoning, PSexpression at the cell surface does not indicate apoptosis unless a dyeexclusion assay has been conducted to establish cell membrane integrity(Vermes et al., 1995).

Nonetheless, the body of literature prior to the present invention doesseem to indicate that the appearance of PS on the outer surface of acell identifies an apoptotic cell and signals that cell's ingestion(Hampton et al., 1996; WO 95/27903). Hampton et al. (1996) concludedthat while an elevation of intracellular Ca²⁺ was an ineffective triggerof apoptosis in the cells investigated, extracellular Ca²⁺ was requiredfor efficient PS exposure during apoptosis. In contrast, the proposal ofMartin et al. (1995) that activation of an inside-outside PS translocaseis an early widespread event during apoptosis would seem to require atleast some intracellular Ca²⁺ (Zhou et al., 1997; Zhao et al., 1998).

Blankenberg et al. (1998) very recently reported that annexin V, anendogenous human protein with a high affinity for PS, can be used toconcentrate at sites of apoptotic cell death in vivo. Radiolabeledannexin V localized to sites of apoptosis in three models, includingacute cardiac allograft rejection (Blankenberg et al., 1998). Stainingof cardiac allografts for exogenously administered annexin V revealedmyocytes at the periphery of mononuclear infiltrates, of which only afew demonstrated positive apoptotic nuclei.

Finally, the transbilayer movement of phospholipids in the plasmamembrane has even been analyzed in ram sperm cells, where the existenceof a transverse segregation of phospholipids has been implicated in thefertilization process (Müller et al., 1994). Phospholipid asymmetry hasthus been receiving increasing attention, although a clear understandingof this phenomenon, or its relationship to health or disease, has notbeen realized.

Irrespective of the confusing state of the art regardingaminophospholipid biology, the present inventors discovered, incontrolled in vivo studies, that aminophospholipids, such as PS and PE,were specific markers of tumor blood vessels. This is surprising inlight of the earlier studies of aminophospholipid function, particularlythose indicating that the cell surface expression of PS is accompaniedby binding of circulating cells, such as T cells (Qu et al., 1996),macrophages (Connor et al., 1989), monocytes (Utsugi et al., 1991) orphagocytes (Zwaal et al., 1989; Williamson and Schlegel, 1994) and is amarker of apoptopic cells (Hampton et al., 1996; Martin et al., 1995;Zhou et al., 1997; Zhao et al., 1998).

Thus, prior to this invention, the possibility of usingaminophospholipids as targetable markers of any disease, let alone oftumor vasculature, would be unlikely to be contemplated, due to theperceived masking of these molecules by the binding of one or more celltypes. In fact, speculative suggestions have concerned the disruption ofPS-cellular interactions, such as in preventing leukocyte binding, aninitial event in atherosclerosis (Qu et al., 1996).

Other surprising aspects of this discovery are evident in a comparisonto earlier work concerning the shedding of procoagulant microparticlesfrom plasma membranes and the demarcation of cells for phagocytosis (WO97/17084). Zwaal et al. (1989; 1992) and Dachary-Prigent et al. (1996)explained that PS translocation to the plasma membrane is followed byrelease of microparticles, microvesicles or microspheres from the cells.Zwaal et al. (1989) and Williamson and Schlegel (1994) indicated that PSsurface expression prompts clearance by the reticuloendothelial system.In light of these fates of PS-expressing cells, and the variousdocumented bilayer translocase activities (Julien et al., 1995; Zhou etal., 1997; Zhao et al., 1998), it is surprising that cell surfaceaminophospholipids such as PS and PE can form static and stable enoughmarkers to allow antibody localization and binding.

Prior to the present invention, there was mounting evidence that surfacePS appears as part of the apoptopic process, marking cells for rapiddestruction (Hampton et al., 1996; Martin et al., 1995). Therefore,although reasonable for use as a diagnostic marker for certain diseasestates, such as graft rejection (Blankenberg et al., 1998), theapparently limited life time of surface PS would also advise against itsuse as a viable marker for targeting in therapeutic intervention.

Nonetheless, the present study did indeed discover aminophospholipids tobe markers of tumor vascular endothelial cells suitable for targeting.After postulating that PS expression was necessary for VCAM coaguligandaction, the presence of PS on tumor blood vessels, but normal vessels,was demonstrated in vivo. The in vivo observations allowed the inventorsto explain the safety and effectiveness of the anti-VCAM coaguligands.This is due to the requirement for coincident expression of a targetedmarker (e.g., VCAM) and PS on tumor endothelium. Even if the targetmolecule is present on endothelium in normal or pathological conditions,thrombosis will not result if surface PS expression is lacking.

The value of the present invention is not limited to explainingcoaguligand action, nor to the surprising development of naked antibodytherapies (provisional application Ser. Nos. 60/092,672 and 60/110,608,each incorporated herein by reference). In fact, the present discoverieshave allowed the inventors to show, for the first time, that PStranslocation in endothelial cells can occur without significant celldamage or cell death (Example XIV). In the inventors' new model of tumorbiology, the translocation of PS to the surface of tumor blood vesselendothelial cells occurs, at least in a significant part, independentlyof apoptopic or other cell-death mechanisms. Thus, PS surface expressionin the tumor environment is not a consequence of cell death, nor does ittrigger immediate cell destruction. This is of fundamental importanceand represents a breakthrough in the scientific understanding of PSbiology, membrane translocation, cell signaling and apoptosis pathways.

The separation of endothelial cell PS translocation from apoptosis(Example XIV) is also integral to methods of therapeutic interventionbased upon PS surface expression. Should PS translocation to the outermembrane in tumor vascular endothelial cells occur only in dying cells,or should it inevitably trigger cell death, then the PS marker would notlikely be sufficiently available to serve as a target for the deliveryof therapeutic agents. That is not to say that PS expression on certaintumor vascular endothelial cells is not transient, and that turnover andcell death do not occur in this endothelial cell population, but thefinding that significant stable PS expression can be achieved withoutcell death is a landmark discovery important to various fields ofbiology and to the new targeted therapeutics described below.

D. Aminophospholipid-Targeted Therapeutics

The in vivo aminophospholipid tumor vasculature expression studiesfurther support the use of coaguligands directed against previouslyidentified tumor vasculature markers, e.g., VCAM-1 and E-selectin, asselective thrombotic agents for the treatment of solid tumors. However,these observations also led the inventors to develop additional tumortreatment methods. For example, naked or unconjugated antibodies againstaminophospholipid components were surprisingly found to be capable ofspecifically inducing tumor blood vessel destruction and tumor necrosisin vivo in the absence of additional effector moieties. Such uses aredisclosed and claimed in first and second provisional application Ser.Nos. 60/092,672 (filed Jul. 13, 1998) and 60/110,608 (filed Dec. 2,1998) and in co-filed U.S. and PCT patent application Ser. Nos.09/351,543, 09/351,862 and PCT/US99/15600, each specificallyincorporated herein by reference.

The studies of first and second provisional application Ser. Nos.60/092,672 (filed Jul. 13, 1998) and 60/110,608 (filed Dec. 2, 1998) arein contrast to those recently reported by Nakamura et al. (1998). Theseauthors analyzed antibody fractions from patients with lupusanticoagulant (LAC), a disorder associated with arterial and venousthrombosis, thrombocytopenia, and recurrent fetal loss. Plasma with LACactivity was initially reported to induce apoptosis in endothelial cells(Nakamura et al., 1994). The apoptotic activities of LAC antisera werethen reported to be localized in an annexin V-binding antibody fractionin 10/10 patients studied (Nakamura et al., 1998). As annexin binds toPS, the apparent ability of anti-annexin antibodies to induce apoptosiswould be the opposite of the ability of an anti-PS antibody to induceapoptosis.

The ability of LAC antibody fractions to induce apoptosis was furtherreported to be inhibited by preincubation with annexin V (Nakamura etal., 1998). In contrast, removal of anti-phospholipid antibodies fromthe patients' IgG fractions with phospholipid liposomes did not abolishthe apoptosis-inducing activities or annexin V binding (Nakamura et al.,1998). These results reasonably implied that patients with LAC oftenhave antibodies that do not bind phospholipids and yet are responsiblefor the induction of apoptosis in endothelial cells (Nakamura et al.,1998).

Without needing to equate the Nakamura et al. (1998) LAC data with theinventors' observations from in vivo studies of tumors and tumorvasculature, due to the evidently disparate nature of these clinicalconditions, the inventors nonetheless have certain unifying theories.Nakamura et al. (1998) attempted to remove anti-phospholipid antibodiesfrom patients' antisera using phospholipid liposomes, and observed thatthis did not abolish the apoptosis-inducing activity. These results ledNakamura et al. (1998) to conclude that the anti-phospholipidsantibodies cannot be responsible for apoptopic activity. However, thepresent inventors now have the insight to suggest that the incubationwith phospholipid liposomes may not have removed the anti-phospholipidsantibodies from the antisera, as phospholipids are antigenically neutralin bilayer and liposomal form, and largely only bind antibodies inhexagonal form (Rauch et al., 1986; Rauch and Janoff, 1990; Berard etal., 1993; each incorporated herein by reference) or in association withmembrane proteins. Thus, anti-phospholipids antibodies may remain in theLAC antisera and may cause, or contribute to, the observed apoptopicactivity.

The invention disclosed herein is directed to the use ofaminophospholipids as targets for anti-tumor vasculature immunotoxinand/or coaguligand therapy. Although the identification of anyadditional target to allow specific tumor vessel localization invascular targeting therapies is valuable, the present discovery ofaminophospholipids as suitable targets is particularly important as itbrings another entire group of targets into the picture: lipids ratherthan the proteins previously preferred. The aminophospholipid discoveryis also functionally significant as it allows therapeutic agents to bedelivered into even more intimate contact with the target cell membrane,rather than binding to a protein complex more distant from the membrane.

One of the most surprising aspects of the present discovery is that PSexpression on intact tumor-associated endothelial cells is sufficentlystable to allow targeting. The present in vivo and in vitro datadefinitively show that PS is expressed on viable tumor-associatedendothelial cells with normal morphology and intact cytoskeletons. As PSexpression is not limited to cells undergoing cell death or about toenter an apoptopic pathway, targeting with diagnostic and therapeuticagents is both practicable and surprising (given that PS expression wasthought to be associated only with cell destruction).

A precise molecular understanding of exactly how and whyaminophospholipid-targeted therapeutic agents are suitable for use intumor treatment is not necessary in order to practice the presentinvention. Given that the administration of aminophospholipid-directedtherapeutic agents is herein shown to advantageously result in specificanti-tumor effects in vivo, the invention can be utilized irrespectiveof the molecular mechanisms that underlie the aminophospholipidexpression in tumor vasculature.

However, it is interesting to note that a review of the scientificliterature to date reveals features that argue against the presentsurprisingly effective uses, and even proposes directly opposite usesfor distinct aminophospholipid binding agent-conjugates. For example,annexin, a phosphatidylserine binding protein, has itself been proposedfor use as an anticoagulant (WO 91/07187; U.S. Pat. No. 5,296,467; eachincorporated herein by reference). This use of annexin was said to bebased upon the inhibition of the procoagulant activity of tumor cells(WO 91/07187).

Even more telling is the disclosure of U.S. Pat. No. 5,632,986 which, incomplete contrast to the present invention, proposes the use of annexinas a conjugate with compounds that lyse thrombi, or precursors of suchthrombolytic compounds. The referenced combination of anaminophospholipid binding protein, annexin, with a lytic agent is,evidently, the opposite of the present invention, which concerns thecombination of annexin and other aminophospholipid binding proteins withagents that induce thrombosis, either directly or indirectly.

In the preparation of both immunotoxins and coaguligands based uponaminophospholipid binding agents and antibodies, recombinant expressionmay be employed to create a fusion protein, as is known to those ofskill in the art and further disclosed herein. Equally, immunotoxins andcoaguligands may be generated using avidin:biotin bridges or any of thechemical conjugation and cross-linker technologies, mostly developed inreference to antibody conjugates. Therefore, any of the followingaminophospholipid binding proteins and ligands may be conjugated to atoxin or coagulant in the same manner as used for antibody conjugates,described herein.

D1. Aminophospholipid Binding Proteins

In addition to antibodies (see below), aminophospholipid binding ligandsor binding proteins may be used in the therapeutic agent-targeting agentconstructs of the present invention. Naturally occurring proteins areknown that bind to both phosphatidylethanolamine and phosphatidylserinewith specificity.

A series of studies by Sugi and McIntyre revealed that kininogens canbind to membrane-exposed PE, at least in platelets (Sugi and McIntyre1995; 1996a; 1996b; each incorporated herein by reference). Kininogensare naturally occurring proteins that normally have anti-thromboticeffects. The present inventors propose that low or high molecular weightkininogens may therefore be attached to therapeutic agents and used inthe delivery of therapeutics to phosphatidylethanolamine, newlydiscovered to be a marker of tumor vasculature.

Various mammalian and human kininogen genes have now been cloned, andsuch genes and proteins can be used in the various recombinant and/orchemical embodiments of the present invention. For example, the completenucleotide and amino acid sequences of the genes and proteins describedin Nakanishi et al., 1983, are incorporated herein by reference for suchpurposes.

Nawa et al. (1983; incorporated herein by reference) reported cDNA andprotein sequences for bovine low molecular weight kininogens. FIG. 2 ofNawa et al. (1983) is specifically incorporated herein by reference forpurposes of providing these complete nucleotide and amino acidsequences. Kitamura et al. (1983; incorporated herein by reference) thenreported that a single gene encodes the bovine high molecular weight andlow molecular weight kininogens. FIG. 2 of Kitamura et al. (1983) isagain incorporated herein by reference to provide the referenced geneand protein sequences. Kitamura et al. (1987) is also specificallyincorporated herein by reference for purposes of providing furtherinformation concerning the bovine, rat and human kininogens, includinglow molecular weight, high molecular weight and T-kininogens.

Preferred high and low molecular weight kininogens for use in theseaspects of the invention will be the human genes and proteins, asdescribed by Takagaki et al. (1985), Kitamura et al. (1985) andKellermann et al. (1986), each incorporated herein by reference. Each ofFIG. 2 and FIG. 3 of Takagaki et al. (1985) are specificallyincorporated herein by reference to provide the complete nucleotide andamino acid sequences of human low and high molecular weightprekininogens, respectively. FIGS. 1 and 8 of the protein analysis paperof Kellermann et al. (1986) are similarly incorporated herein.

Kitamura et al. (1985) is also specifically incorporated herein byreference for purposes of providing further information regarding thestructural organization of the human kininogen gene, as may be used,e.g., to design particular expression constructs for use herewith.Kitamura et al. (1988) is further incorporated by reference for purposesof providing detailed information regarding the cloning of cDNAs andgenomic kininogens, such that any desired kininogen may be cloned.

In addition to the T-kininogens described by Kitamura et al. (1987;incorporated herein by reference), Anderson et al. (1989) is alsospecifically incorporated herein by reference for purposes of providingthe gene and protein sequences of T-kininogen. FIG. 3 of Anderson et al.(1989) is specifically incorporated.

Other phosphatidylethanolamine binding proteins are known that can beused in such embodiments. A number of studies, particularly by Jones andHall, and Bernier and Jolles, have concerned the purification,characterization and cloning of phosphatidylethanolamine bindingproteins. For example, Bernier and Jolles (1984; incorporated herein byreference) first reported the purification and characterization of abasic ˜23 kDa cytosolic protein from bovine brain that was latercharacterized as a phosphatidylethanolamine-binding protein (Bernier etal., 1986; incorporated herein by reference). Schoentgen et al. (1987;incorporated herein by reference) reported the complete amino acidsequence of this bovine protein, then shown to be 21 kDa. FIG. 2 ofSchoentgen et al. (1987) is specifically incorporated herein byreference for purposes of providing the complete amino acid sequence ofthis bovine phosphatidylethanolamine binding protein.

Jones and Hall (1991; incorporated herein by reference) later purifiedand partially sequenced a ˜23 kDa protein from rat sperm plasmamembranes that showed sequence similarity and phospholipid bindingproperties similar to the bovine brain cytosolic protein of Bernier andJolles (Bernier and Jolles, 1984; Bernier et al., 1986; Schoentgen etal., 1987). The rat 23 kDa protein of Jones and Hall (1991; incorporatedherein by reference) also showed selective affinity forphosphatidylethanolamine (Kd=1.6×10⁻⁵ M).

Perry et al. (1994; incorporated herein by reference) then cloned andsequenced rat and monkey versions of the phosphatidylethanolaminebinding protein of Jones and Hall (1991). FIGS. 4, 5 and 6 of Perry etal. (1994) are specifically incorporated herein by reference forpurposes of providing the complete DNA and amino acid sequences of therat and monkey phosphatidylethanolamine binding proteins, and comparisonto the bovine protein sequence. Any of the foregoing mammalianphosphatidylethanolamine binding proteins, or their human counterparts,may be attached to therapeutic agents and used in the present invention.These mammalian sequences have EMBL Nucleotide Sequence DatabaseAccession Numbers X71873 (rat) and X73137 (monkey), and are eachincorporated herein by reference.

To counterpart human phosphatidylethanolamine binding protein has alsobeen cloned (Hori et al., 1994; incorporated herein by reference). BothFIG. 1 of Hori et al. (1994) and GenBank, EMBL and DDBJ Accession NumberD16111 are incorporated herein by reference for purposes of providingthe complete DNA and amino acid sequences of the humanphosphatidylethanolamine binding proteins. The mammalian and humansequences, as incorporated herein, may be employed in well-knownexpression techniques, either to express the proteins themselves ortherapeutic agent-fusions thereof. Phosphatidylethanolamine bindingproteins and genes from other sources, such as yeast, Drosophila,simian, T. canis and O. volvulus may also be employed in theseembodiments (Gems et al., 1995; incorporated herein by reference).

Variant, mutant or second generation phosphatidylethanolamine bindingprotein nucleic acids may also be readily prepared by standard molecularbiological techniques, and may optionally be characterized ashybridizing to any of the phosphatidylethanolamine binding proteinnucleotide sequences set forth in any one or more of Nakanishi et al.(1983); Nawa et al. (1983); Kitamura et al. (1983; 1985; 1987; 1988);Takagaki et al. (1985); Kellermann et al. (1986); Anderson et al.(1989); Bernier and Jolles (1984); Bernier et al. (1986); Schoentgen etal. (1987); Jones and Hall (1991); Perry et al. (1994); and Hori et al.(1994); each incorporated herein by reference. Exemplary suitablehybridization conditions include hybridization in about 7% sodiumdodecyl sulfate (SDS), about 0.5 M NaPO₄, about 1 mM EDTA at about 50°C.; and washing with about 1% SDS at about 42° C.

In addition to the foregoing phosphatidylethanolamine binding proteinsor “ligands”, naturally occurring proteins exist that specifically bindphosphatidylserine. Preferred amongst these are annexins (sometimesspelt “annexines”), a group of calcium-dependent phospholipid bindingproteins. At least nine members of the annexin family have beenidentified in mammalian tissues (Annexin I through Annexin IX). Mostpreferred amongst these is annexin V (also known as PAP-I).

U.S. Pat. No. 5,658,877, incorporated herein by reference, describesAnnexin I, effective amounts of Annexin I and pharmaceuticalcompositions thereof. Also described are methods of treating an animalto prevent or alleviate the adverse effects of endotoxin in the lungthat comprise administering into the airway of an animal a safe amountof 33 kDa Annexin I fragment.

Annexin V contains one free sulfhydryl group and does not have anyattached carbohydrate chains. The primary structure of annexin V deducedfrom the cDNA sequence shows that annexin V comprises four internalrepeating units (U.S. Pat. No. 4,937,324; incorporated herein byreference).

U.S. Pat. No. 5,296,467 and WO 91/07187 are also each incorporatedherein by reference as they provide pharmaceutical compositionscomprising ‘annexine’ (annexin). Although proposed for use asanticoagulants, the annexins of U.S. Pat. No. 5,296,467 and WO 91/07187may now be used as part of the conjugates of the present invention.

WO 91/07187 provides natural, synthetic or genetically preparedderivatives and analogues of ‘annexine’ (annexin), which may now be usedin the conjugates of the present invention. Particular annexins areprovided of 320 amino acids, containing variant amino acids and,optionally, a disulphide bridge between the 316-Cys and the 2-Ala.

U.S. Pat. No. 5,296,467 is incorporated herein by reference in itsentirety, including all figures and sequences, for purposes of evenfurther describing annexins and pharmaceutical compositions thereof.U.S. Pat. No. 5,296,467 describes annexin cloning, recombinantexpression and preparation. Aggregates of two or more annexines, e.g.,linked by disulfide bonds between one or more cysteine groups on therespective annexine, are also disclosed. Yet a further example ofsuitable annexin starting materials is provided by WO 95/27903(incorporated herein by reference), which provides annexins for use indetecting apoptotic cells.

WO 97/17084 is also incorporated herein by reference for purposes ofdescribing annexin starting materials for preparing constructs of thepresent invention. WO 97/17084 particularly concerns the use of AnnexinV to alter phosphatidylserine-dependent phagocytosis. It is said thatblocking PS-dependent phagocytosis means that PS-carrying cells undergophagocytosis by other pathways, leading to greater immune responses,such that Annexin V may be used as an adjuvant to increaseimmunogenicity of vaccines. The treatment of sickle cell anemia andmalaria is also described. WO 97/17084 also provides certain expressionvector systems that may be adapted for use herein.

To the extent that they clearly describe appropriate annexin startingmaterials for preparing therapeutic constructs of the present invention,each of the diagnostic approaches of U.S. Pat. No. 5,627,036; WO95/19791; WO 95/27903; WO 95/34315; WO 96/17618; and WO 98/04294; arealso specifically incorporated herein by reference. Various of thesedocuments also concern recombinant expression vectors useful foradaptation into the present invention.

Although totally counter-intuitive prior to the present invention, theannexin conjugation technology of U.S. Pat. No. 5,632,986 may now beadapted for use in the present tumor treatment methods. U.S. Pat. No.5,632,986 (incorporated herein by reference) provides annexin conjugatesusing compounds that lyse thrombi, or precursors of such compounds.Annexin-plasminogen activator conjugates and annexin-urokinaseconjugates were particularly provided for thrombolysis and for treatingdisorders resulting from thrombosis. By switching the thrombolyticcompounds of U.S. Pat. No. 5,632,986 for the toxic and coagulativecompounds disclosed herein, the basic conjugate technology of U.S. Pat.No. 5,632,986 can be easily adapted for use in the present invention.

U.S. Pat. No. 5,632,986 is thus provided for purposes of furtherdescribing annexin isolation from tissue extracts (U.S. Pat. No.4,937,324; also incorporated herein by reference) and annexin productionby recombinant methods. Each of the cDNA clones and expression vectorsof U.S. Pat. No. 5,632,986 are thus specifically incorporated herein byreference.

U.S. Pat. No. 5,632,986 is also specifically incorporated herein byreference for purposes of further describing mutants and variants of theannexin molecule that are subdivided or altered at one or more aminoacid residues so long as the phospholipid binding capability is notreduced substantially. Appropriate annexins for use in the presentinvention can thus be truncated, for example, to include one or moredomains or contain fewer amino acid residues than the native protein, orcan contain substituted amino acids. Any changes are acceptable withinthe scope of the invention so long as the mutein or second generationannexin molecule does not contain substantially lower affinity foraminophospholipid. Such guidance can also be applied tophosphatidylethanolamine binding proteins.

Second generation, variant and mutant annexin-encoding nucleic acids mayalso be readily prepared by standard molecular biological techniques,and may optionally be characterized as hybridizing to any of theforegoing annexin-encoding nucleic acid sequences under hybridizationconditions such as those including hybridization in about 7% sodiumdodecyl sulfate (SDS), about 0.5 M NaPO₄, about 1 mM EDTA at about 50°C.; and washing with about 1% SDS at about 42° C.

The chemical cross-linking of annexins and other agents is alsodescribed in U.S. Pat. No. 5,632,986, incorporated herein by reference.All such techniques can be adapted for use herewith simply bysubstituting the thrombolytic agents for those described herein.Aliphatic diamines; succinimide esters; hetero-bifunctional couplingreagents, such as SPDP; maleimide compounds; linkers with spacers; andthe like, may thus be used.

U.S. Pat. No. 5,632,986 is yet further specifically incorporated hereinby reference for purposes of describing the recombinant production ofannexin-containing conjugates. Appropriate nucleic acid sequences arethus joined to produce chimeric coding sequences that, in turn, producechimeric proteins. Exemplary expression vectors are said to be pKK233-2(E. coli), DPOT (yeast) and pDSP1.1BGH (mammalian). Such teaching issupplemented by further information provided herein.

D2. Biologically Functional Equivalents

Equivalents, or even improvements, of aminophospholipid binding proteinscan now be made, generally using the materials provided above as astarting point. Modifications and changes may be made in the structureof an aminophospholipid binding protein and still obtain a moleculehaving like or otherwise desirable characteristics. For example, certainamino acids may substituted for other amino acids in a protein structurewithout appreciable loss of interactive binding capacity, such as,binding to the aminophospholipids, PS and PE. These considerations alsoapply to toxins and coagulants.

Since it is the interactive capacity and nature of a protein thatdefines that protein's biological functional activity, certain aminoacid sequence substitutions can be made in a protein sequence (or ofcourse, the underlying DNA sequence) and nevertheless obtain a proteinwith like (agonistic) properties. It is thus contemplated that variouschanges may be made in the sequence of known aminophospholipid bindingproteins or peptides (or underlying DNA sequences) without appreciableloss of their biological utility or activity. Biological functionalequivalents made from mutating an underlying DNA sequence can be madeusing the codon information provided herein in Table A, and thesupporting technical details on site-specific mutagenesis.

It also is well understood by the skilled artisan that, inherent in thedefinition of a “biologically functional equivalent” protein or peptide,is the concept that there is a limit to the number of changes that maybe made within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity.Biologically functional equivalent proteins and peptides are thusdefined herein as those proteins and peptides in which certain, not mostor all, of the amino acids may be substituted. Of course, a plurality ofdistinct proteins/peptides with different substitutions may easily bemade and used in accordance with the invention.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

In making more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte and Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is thus understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent protein. As detailed in U.S. Pat. No. 4,554,101 (incorporatedherein by reference), the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those which are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

D3. Toxic and Anti-Cellular Agents

For certain applications, the therapeutic agents will be cytotoxic orpharmacological agents, particularly cytotoxic, cytostatic,anti-cellular or anti-angiogenic agents having the ability to kill orsuppress the growth or cell division of endothelial cells. In general,these aspects of the invention contemplate the use of anypharmacological agent that can be conjugated to a targeting agent, anddelivered in active form to the targeted endothelium.

Exemplary anti-cellular agents include chemotherapeutic agents, as wellas cytotoxins. Chemotherapeutic agents that may be used include:hormones, such as steroids; anti-metabolites, such as cytosinearabinoside, fluorouracil, methotrexate or aminopterin; anthracyclines;mitomycin C; vinca alkaloids; demecolcine; etoposide; mithramycin;anti-tumor alkylating agents, such as chlorambucil or melphalan. Otherembodiments may include agents such as cytokines. Basically, anyanti-cellular agent may be used, so long as it can be successfullyconjugated to, or associated with, a targeting agent or antibody in amanner that will allow its targeting, internalization, release and/orpresentation to blood components at the site of the targeted endothelialcells.

There may be circumstances, such as when the target antigen does notinternalize by a route consistent with efficient intoxication by thetoxic compound, where one will desire to target chemotherapeutic agents,such as anti-tumor drugs, cytokines, antimetabolites, alkylating agents,hormones, and the like. A variety of chemotherapeutic and otherpharmacological agents have now been successfully conjugated toantibodies and shown to function pharmacologically, includingdoxorubicin, daunomycin, methotrexate, vinblastine, neocarzinostatin,macromycin, trenimon and α-amanitin.

In other circumstances, any potential side-effects from cytotoxin-basedtherapy may be eliminated by the use of DNA synthesis inhibitors, suchas daunorubicin, doxorubicin, adriamycin, and the like. These agents aretherefore preferred examples of anti-cellular agents for use in thepresent invention. In terms of cytostatic agents, such compoundsgenerally disturb the natural cell cycle of a target cell, preferably sothat the cell is taken out of the cell cycle. Exemplary cytostaticagents include.

A wide variety of cytotoxic agents are known that may be conjugated toanti-aminophospholipid antibodies or binding ligands. Examples includenumerous useful plant-, fungus- or bacteria-derived toxins, which, byway of example, include various A chain toxins, particularly ricin Achain; ribosome inactivating proteins, such as saporin or gelonin;α-sarcin; aspergillin; restrictocin; ribonucleases, such as placentalribonuclease; diphtheria toxin; and pseudomonas exotoxin, to name just afew.

Of the toxins, ricin A chains are preferred. The most preferred toxinmoiety for use herewith is toxin A chain that has been treated to modifyor remove carbohydrate residues, so-called deglycosylated A chain (dgA).Deglycosylated ricin A chain is preferred because of its extremepotency, longer half-life, and because it is economically feasible tomanufacture it in a clinical grade and scale.

It may be desirable from a pharmacological standpoint to employ thesmallest molecule possible that nevertheless provides an appropriatebiological response. One may thus desire to employ smaller A chainpeptides that will provide an adequate anti-cellular response. To thisend, it has been discovered that ricin A chain may be “truncated” by theremoval of 30 N-terminal amino acids by Nagarase (Sigma), and stillretain an adequate toxin activity. It is proposed that where desired,this truncated A chain may be employed in conjugates in accordance withthe invention.

Alternatively, one may find that the application of recombinant DNAtechnology to the toxin A chain moiety will provide additional benefitsin accordance the invention. In that the cloning and expression ofbiologically active ricin A chain has been achieved, it is now possibleto identify and prepare smaller or otherwise variant peptides whichnevertheless exhibit an appropriate toxin activity. Moreover, the factthat ricin A chain has now been cloned allows the application ofsite-directed mutagenesis, through which one can readily prepare andscreen for A chain-derived peptides and obtain additional usefulmoieties for use in connection with the present invention.

Other agents for use in immunoconjugate targeting of PS expressed ontumor vasculature are the angiopoietins. The angiopoietins, like themembers of the VEGF family, are growth factors largely specific forvascular endothelium (Davis and Yancopoulos, 1999; Holash et al., 1999;incorporated herein by reference). The angiopoietins first describedwere a naturally occurring agonist, angiopoietin-1 (Ang-1; SEQ ID NO:1and SEQ ID NO:2), and a naturally occurring antagonist, angiopoietin-2(Ang-2; SEQ ID NO:3 and SEQ ID NO:4), both of which act by means of theendothelial cell tyrosine kinase receptor, Tie2.

Two new angiopoietins, angiopoietin-3 (mouse) and angiopoietin-4 (human)have also been identified (Valenzuela et al., 1999). Angiopoietin-3appears to act as an antagonist, whereas angiopoietin-4 appears tofunction as an agonist (Valenzuela et al., 1999). A protein termedangiopoietin-3 was also cloned from human heart and reported not to havemitogenic effects on endothelial cells (Kim et al., 1999).

Whereas VEGF is necessary for the early stages of vascular development,angiopoietin-1 is generally required for the later stages of vascularremodeling. Angiopoietin-1 is thus a maturation or stabilization factor,which converts immature vessels to mature vessels.

Angiopoietin-1 has been shown to augment revascularization in ischemictissue (Shyu et al., 1998) and to increase the survival of vascularnetworks exposed to either VEGF or a form of aFGF (Papapetropoulos etal., 1999). These authors also showed that angiopoietin-1 preventsapoptotic death in HUVEC triggered by withdrawal of the same form ofaFGF (Papapetropoulos et al., 1999). Such data are consistent with thedirect role of angiopoietin-1 on human endothelial cells and itsinteraction with other angiogenic molecules to stabilize vascularstructures by promoting the survival of differentiated endothelialcells.

Of the angiopoietins, angiopoietin-2 is a preferred agent for use inPS-targeted therapy, particularly in tumors with low VEGF levels and/orin combination with VEGF inhibition. Angiopoietin-2 is also a ligand forTie2, but generally counteracts blood vessel maturation/stabilitymediated by angiopoietin-1. It is thus an antagonist of angiopoietin-1,and acts to disturb capillary structure. However, as angiopoietin-2renders endothelial cells responsive to angiogenic stimuli, it caninitiate neovascularization in combination with other appropriatesignals, particularly VEGF (Asahara et al., 1998; Holash et al., 1999;incorporated herein by reference).

In the absence of another angiogenic signal, angiopoietin-2 causesvessels to destabilize and become immature. In the presence of astimulus, such as VEGF, angiopoietin-2 promotes angiogenesis. Indeed,the angiogenic effects of a number of regulators are believed to beachieved, at least in part, through the regulation of an autocrine loopof angiopoietin-2 activity in microvascular endothelial cells (Mandriotaand Pepper, 1998).

Angiopoietin-2 expression in tumor tissue has been reported (Tanaka etal., 1999), where it presumably acts in combination with VEGF to promoteangiogenesis (Stratmann et al., 1998). However, as angiopoietin-2provides a negative signal when VEGF is low or absent, provision ofangiopoietin-2 can be a useful therapeutic approach. In addition totumor-targeted forms, angiopoietin-2 can also be administered as aprotein or gene therapy therapeutic (see combination therapies describedherein). Fusion proteins of angiopoietins are also envisioned for use inthis invention, such as the stable Ang-1-Ang-2 fusion protein includedherein as SEQ ID NO:5.

D4. Coagulation Factors

The antibody and ligand targeting agents of the invention may be linkedto a component that is capable of directly or indirectly stimulatingcoagulation, to form a coaguligand. Here, the targeting agents may bedirectly linked to the coagulant or coagulation factor, or may be linkedto a second binding region that binds and then releases the coagulant orcoagulation factor. As used herein, the terms “coagulant” and“coagulation factor” are each used to refer to a component that iscapable of directly or indirectly stimulating coagulation underappropriate conditions, preferably when provided to a specific in vivoenvironment, such as the tumor vasculature.

Preferred coagulation factors are Tissue Factor compositions, such astruncated TF (tTF), dimeric, multimeric and mutant TF molecules.“Truncated TF” (tTF) refers to TF constructs that are renderedmembrane-binding deficient by removal of sufficient amino acid sequencesto effect this change in property. A “sufficient amount” in this contextis an amount of transmembrane amino acid sequence originally sufficientto enter the TF molecule in the membrane, or otherwise mediatefunctional membrane binding of the TF protein. The removal of such a“sufficient amount of transmembrane spanning sequence” therefore createsa truncated Tissue Factor protein or polypeptide deficient inphospholipid membrane binding capacity, such that the protein issubstantially a soluble protein that does not significantly bind tophospholipid membranes. Truncated TF thus substantially fails to convertFactor VII to Factor VIIa in a standard TF assay, and yet retainsso-called catalytic activity including activating Factor X in thepresence of Factor VIIa.

U.S. Pat. No. 5,504,067 is specifically incorporated herein by referencefor the purposes of further describing such truncated Tissue Factorproteins. Preferably, the Tissue Factors for use in these aspects of thepresent invention will generally lack the transmembrane and cytosolicregions (amino acids 220–263) of the protein. However, there is no needfor the truncated TF molecules to be limited to molecules of the exactlength of 219 amino acids.

Tissue Factor compositions may also be useful as dimers. Any of thetruncated, mutated or other Tissue Factor constructs may be prepared ina dimeric form for use in the present invention. As will be known tothose of ordinary skill in the art, such TF dimers may be prepared byemploying the standard techniques of molecular biology and recombinantexpression, in which two coding regions are prepared in-frame andexpressed from an expression vector. Equally, various chemicalconjugation technologies may be employed in connection with thepreparation of TF dimers. The individual TF monomers may be derivatizedprior to conjugation. All such techniques would be readily known tothose of skill in the art.

If desired, the Tissue Factor dimers or multimers may be joined via abiologically-releasable bond, such as a selectively-cleavable linker oramino acid sequence. For example, peptide linkers that include acleavage site for an enzyme preferentially located or active within atumor environment are contemplated. Exemplary forms of such peptidelinkers are those that are cleaved by urokinase, plasmin, thrombin,Factor IXa, Factor Xa, or a metalloproteinase, such as collagenase,gelatinase or stromelysin.

In certain embodiments, the Tissue Factor dimers may further comprise ahindered hydrophobic membrane insertion moiety, to later encourage thefunctional association of the Tissue Factor with the phospholipidmembrane, but only under certain defined conditions. As described in thecontext of the truncated Tissue Factors, hydrophobicmembrane-association sequences are generally stretches of amino acidsthat promote association with the phospholipid environment due to theirhydrophobic nature. Equally, fatty acids may be used to provide thepotential membrane insertion moiety.

Such membrane insertion sequences may be located either at theN-terminus or the C-terminus of the TF molecule, or generally appendedat any other point of the molecule so long as their attachment theretodoes not hinder the functional properties of the TF construct. Theintent of the hindered insertion moiety is that it remainsnon-functional until the TF construct localizes within the tumorenvironment, and allows the hydrophobic appendage to become accessibleand even further promote physical association with the membrane. Again,it is contemplated that biologically-releasable bonds andselectively-cleavable sequences will be particularly useful in thisregard, with the bond or sequence only being cleaved or otherwisemodified upon localization within the tumor environment and exposure toparticular enzymes or other bioactive molecules.

In other embodiments, the tTF constructs may be multimeric or polymeric.In this context a “polymeric construct” contains 3 or more Tissue Factorconstructs. A “multimeric or polymeric TF construct” is a construct thatcomprises a first TF molecule or derivative operatively attached to atleast a second and a third TF molecule or derivative. The multimers maycomprise between about 3 and about 20 such TF molecules. The individualTF units within the multimers or polymers may also be linked byselectively-cleavable peptide linkers or other biological-releasablebonds as desired. Again, as with the TF dimers discussed above, theconstructs may be readily made using either recombinant manipulation andexpression or using standard synthetic chemistry.

Even further TF constructs useful in context of the present inventionare those mutants deficient in the ability to activate Factor VII. Such“Factor VII activation mutants” are generally defined herein as TFmutants that bind functional Factor VII/VIIa, proteolytically activateFactor X, but are substantially free from the ability to proteolyticallyactivate Factor VII. Accordingly, such constructs are TF mutants thatlack Factor VII activation activity.

The ability of such Factor VII activation mutants to function inpromoting tumor-specific coagulation is based upon their specificdelivery to the tumor vasculature, and the presence of Factor VIIa atlow levels in plasma. Upon administration of such a Factor VIIactivation mutant-targeting agent conjugate, the mutant will belocalized within the vasculature of a vascularized tumor. Prior tolocalization, the TF mutant would be generally unable to promotecoagulation in any other body sites, on the basis of its inability toconvert Factor VII to Factor VIIa. However, upon localization andaccumulation within the tumor region, the mutant will then encountersufficient Factor VIIa from the plasma in order to initiate theextrinsic coagulation pathway, leading to tumor-specific thrombosis.Exogenous Factor VIIa could also be administered to the patient.

Any one or more of a variety of Factor VII activation mutants may beprepared and used in connection with the present invention. There is asignificant amount of scientific knowledge concerning the recognitionsites on the TF molecule for Factor VII/VIIa. It will thus be understoodthat the Factor VII activation region generally lies between about aminoacid 157 and about amino acid 167 of the TF molecule. However, it iscontemplated that residues outside this region may also prove to berelevant to the Factor VII activating activity, and one may thereforeconsider introducing mutations into any one or more of the residuesgenerally located between about amino acid 106 and about amino acid 209of the TF sequence (WO 94/07515; WO 94/28017; each incorporated hereinby reference).

A variety of other coagulation factors may be used in connection withthe present invention, as exemplified by the agents set forth below.Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIa andderivatives, Factor IX/IXa and derivatives, Factor X/Xa and derivatives,Factor XI/XIa and derivatives, Factor XII/XIIa and derivatives, FactorXIII/XIIIa and derivatives, Factor X activator and Factor V activatormay be used in the present invention.

Russell's viper venom Factor X activator is contemplated for use in thisinvention. Monoclonal antibodies specific for the Factor X activatorpresent in Russell's viper venom have also been produced, and could beused to specifically deliver the agent as part of a bispecific bindingligand.

Thromboxane A₂ is formed from endoperoxides by the sequential actions ofthe enzymes cyclooxygenase and thromboxane synthetase in plateletmicrosomes. Thromboxane A₂ is readily generated by platelets and is apotent vasoconstrictor, by virtue of its capacity to produce plateletaggregation. Both thromboxane A₂ and active analogues thereof arecontemplated for use in the present invention.

Thromboxane synthase, and other enzymes that synthesizeplatelet-activating prostaglandins, may also be used as “coagulants” inthe present context. Monoclonal antibodies to, and immunoaffinitypurification of, thromboxane synthase are known; as is the cDNA forhuman thromboxane synthase.

α2-antiplasmin, or α2-plasmin inhibitor, is a proteinase inhibitornaturally present in human plasma that functions to efficiently inhibitthe lysis of fibrin clots induced by plasminogen activator.α2-antiplasmin is a particularly potent inhibitor, and is contemplatedfor use in the present invention.

As the cDNA sequence for α2-antiplasmin is available, recombinantexpression and/or fusion proteins are preferred. Monoclonal antibodiesagainst α2-antiplasmin are also available that may be used in thebispecific binding ligand embodiments of the invention. These antibodiescould both be used to deliver exogenous α2-antiplasmin to the targetsite or to garner endogenous α2-antiplasmin and concentrate it withinthe targeted region.

D5. Fusion Proteins and Recombinant Expression

The therapeutic agent-targeting agent compositions of the presentinvention may be readily prepared as fusion proteins using molecularbiological techniques. The use of recombinant DNA techniques to achievesuch ends is now standard practice to those of skill in the art. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.DNA and RNA synthesis may, additionally, be performed using an automatedsynthesizers (see, for example, the techniques described in Sambrook etal., 1989).

The preparation of such a fusion protein generally entails thepreparation of a first and second DNA coding region and the functionalligation or joining of such regions, in frame, to prepare a singlecoding region that encodes the desired fusion protein. In the presentcontext, the targeting agent DNA sequence will be joined in frame with aDNA sequence encoding a therapeutic agent. It is not generally believedto be particularly relevant which portion of the therapeuticagent-targeting agent is prepared as the N-terminal region or as theC-terminal region.

Once the desired coding region has been produced, an expression vectoris created. Expression vectors contain one or more promoters upstream ofthe inserted DNA regions that act to promote transcription of the DNAand to thus promote expression of the encoded recombinant protein. Thisis the meaning of “recombinant expression”.

To obtain a so-called “recombinant” version of the therapeuticagent-targeting agent protein, it is expressed in a recombinant cell.The engineering of DNA segment(s) for expression in a prokaryotic oreukaryotic system may be performed by techniques generally known tothose of skill in recombinant expression. It is believed that virtuallyany expression system may be employed in the expression of thetherapeutic agent-targeting agent constructs.

Such proteins may be successfully expressed in eukaryotic expressionsystems, e.g., CHO cells, however, it is envisioned that bacterialexpression systems, such as E. coli pQE-60 will be particularly usefulfor the large-scale preparation and subsequent purification of thetherapeutic agent-targeting agent constructs. cDNAs may also beexpressed in bacterial systems, with the encoded proteins beingexpressed as fusions with β-galactosidase, ubiquitin, Schistosomajaponicum glutathione S-transferase, and the like. It is believed thatbacterial expression will have advantages over eukaryotic expression interms of ease of use and quantity of materials obtained thereby.

In terms of microbial expression, U.S. Pat. Nos. 5,583,013; 5,221,619;4,785,420; 4,704,362; and 4,366,246 are incorporated herein by referencefor the purposes of even further supplementing the present disclosure inconnection with the expression of genes in recombinant host cells.

Recombinantly produced therapeutic agent-targeting agent constructs maybe purified and formulated for human administration. Alternatively,nucleic acids encoding the therapeutic agent-targeting agent constructsmay be delivered via gene therapy. Although naked recombinant DNA orplasmids may be employed, the use of liposomes or vectors is preferred.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and to integrate into the host cell genome and express viralgenes stably and efficiently have made them attractive candidates forthe transfer of foreign genes into mammalian cells. Preferred genetherapy vectors for use in the present invention will generally be viralvectors.

Retroviruses have promise as gene delivery vectors due to their abilityto integrate their genes into the host genome, transferring a largeamount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and of being packaged in special cell-lines.Other viruses, such as adenovirus, herpes simplex viruses (HSV),cytomegalovirus (CMV), and adeno-associated virus (AAV), such as thosedescribed by U.S. Pat. No. 5,139,941 (incorporated herein by reference),may also be engineered to serve as vectors for gene transfer.

Although some viruses that can accept foreign genetic material arelimited in the number of nucleotides they can accommodate and in therange of cells they infect, these viruses have been demonstrated tosuccessfully effect gene expression. However, adenoviruses do notintegrate their genetic material into the host genome and therefore donot require host replication for gene expression, making them ideallysuited for rapid, efficient, heterologous gene expression. Techniquesfor preparing replication-defective infective viruses are well known inthe art.

In certain further embodiments, the gene therapy vector will be HSV. Afactor that makes HSV an attractive vector is the size and organizationof the genome. Because HSV is large, incorporation of multiple genes orexpression cassettes is less problematic than in other smaller viralsystems. In addition, the availability of different viral controlsequences with varying performance (e.g., temporal, strength) makes itpossible to control expression to a greater extent than in othersystems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations. HSV also isrelatively easy to manipulate and can be grown to high titers.

Of course, in using viral delivery systems, one will desire to purifythe virion sufficiently to render it essentially free of undesirablecontaminants, such as defective interfering viral particles orendotoxins and other pyrogens such that it will not cause any untowardreactions in the cell, animal or individual receiving the vectorconstruct. A preferred means of purifying the vector involves the use ofbuoyant density gradients, such as cesium chloride gradientcentrifugation.

E. Anti-Aminophospholipid Antibodies and Conjugates

E1. Polyclonal Anti-Aminophospholipid Antibodies

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference). To preparepolyclonal antisera an animal is immunized with an immunogenicaminophospholipid composition, and antisera collected from thatimmunized animal. A wide range of animal species can be used for theproduction of antisera. Typically the animal used for production ofanti-antisera is a rabbit, mouse, rat, hamster, guinea pig or goat.Because of the relatively large blood volume of rabbits, a rabbit is apreferred choice for production of polyclonal antibodies.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer thepresent aminophospholipid immunogen; subcutaneous, intramuscular,intradermal, intravenous, intraperitoneal and intrasplenic. Theproduction of polyclonal antibodies may be monitored by sampling bloodof the immunized animal at various points following immunization. Asecond, booster injection, may also be given. The process of boostingand titering is repeated until a suitable titer is achieved. When adesired titer level is obtained, the immunized animal can be bled andthe serum isolated and stored. The animal can also be used to generatemonoclonal antibodies.

As is well known in the art, the immunogenicity of a particularcomposition can be enhanced by the use of non-specific stimulators ofthe immune response, known as adjuvants. Exemplary adjuvants includecomplete Freund's adjuvant, a non-specific stimulator of the immuneresponse containing killed Mycobacterium tuberculosis; incompleteFreund's adjuvant; and aluminum hydroxide adjuvant.

It may also be desired to boost the host immune system, as may beachieved by associating aminophospholipids with, or couplingaminophospholipids to, a carrier. Exemplary carriers are keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such asovalbumin, mouse serum albumin or rabbit serum albumin can also be usedas carriers.

As is also known in the art, a given composition may vary in itsimmunogenicity. However, the generation of antibodies againstaminophospholipids is not particularly difficult. For example, highlyspecific anti-phosphatidylserine antibodies were raised in rabbitsimmunized by intramuscular injections of phosphatidylserine-containingpolyacrylamide gels and with phosphatidylserine-cytochrome c vesicles(Maneta-Peyret et al., 1988; 1989; each incorporated herein byreference). The use of acrylamide implants enhanced the production ofantibodies (Maneta-Peyret et al., 1988; 1989). Theanti-phosphatidylserine antibodies raised in this manner are able todetect phosphatidylserine in situ on human platelets (Maneta-Peyret etal., 1988). The groups of Inoue, Rote and Rauch have also developedanti-PS and anti-PE antibodies (see below).

E2. Monoclonal Anti-Aminophospholipid Antibodies

Various methods for generating monoclonal antibodies (MAbs) are also nowvery well known in the art. The most standard monoclonal antibodygeneration techniques generally begin along the same lines as those forpreparing polyclonal antibodies (Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference). Apolyclonal antibody response is initiated by immunizing an animal withan immunogenic aminophospholipid composition and, when a desired titerlevel is obtained, the immunized animal can be used to generate MAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with the selected aminophospholipid immunogen composition. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep and frog cells isalso possible. The use of rats may provide certain advantages (Goding,1986, pp. 60–61; incorporated herein by reference), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingaminophospholipid antibodies, specifically B lymphocytes (B cells), areselected for use in the MAb generating protocol. These cells may beobtained from biopsied spleens, tonsils or lymph nodes, or from aperipheral blood sample. Spleen cells and peripheral blood cells arepreferred, the former because they are a rich source ofantibody-producing cells that are in the dividing plasmablast stage, andthe latter because peripheral blood is easily accessible. Often, a panelof animals will have been immunized and the spleen of animal with thehighest antibody titer will be removed and the spleen lymphocytesobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×10⁷ to 2×10⁸lymphocytes.

The anti-aminophospholipid antibody-producing B lymphocytes from theimmunized animal are then fused with cells of an immortal myeloma cell,generally one of the same species as the animal that was immunized.Myeloma cell lines suited for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render then incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65–66, 1986; Campbell, pp. 75–83, 1984;each incorporated herein by reference). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F, 4B210 or one of theabove listed mouse cell lines; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6, are all useful in connection with human cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 4:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976; each incorporated herein by reference),and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, byGefter et al. (1977; incorporated herein by reference). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71–74, 1986; incorporated herein by reference).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁸ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired anti-aminophospholipid reactivity. Theassay should be sensitive, simple and rapid, such as radioimmunoassays,enzyme immunoassays, cytotoxicity assays, plaque assays, dotimmunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual anti-aminophospholipid antibody-producing cell lines, whichclones can then be propagated indefinitely to provide MAbs. The celllines may be exploited for MAb production in two basic ways. A sample ofthe hybridoma can be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion. The injected animal developstumors secreting the specific monoclonal antibody produced by the fusedcell hybrid. The body fluids of the animal, such as serum or ascitesfluid, can then be tapped to provide MAbs in high concentration. Theindividual cell lines could also be cultured in vitro, where the MAbsare naturally secreted into the culture medium from which they can bereadily obtained in high concentrations.

MAbs produced by either means will generally be further purified, e.g.,using filtration, centrifugation and various chromatographic methods,such as HPLC or affinity chromatography, all of which purificationtechniques are well known to those of skill in the art. Thesepurification techniques each involve fractionation to separate thedesired antibody from other components of a mixture. Analytical methodsparticularly suited to the preparation of antibodies include, forexample, protein A-Sepharose and/or protein G-Sepharose chromatography.

Umeda et al. (1989; incorporated herein by reference) reported theeffective production of monoclonal antibodies recognizingstereo-specific epitopes of phosphatidylserine. The Umeda system isbased on the direct immunization of phosphatidylserine into mouse spleenusing a Salmonella-coated aminophospholipid sample (Umeda et al., 1989;incorporated herein by reference). The Umeda protocol gives a highfrequency of anti-PS MAbs, which exhibit three distinct reactivityprofiles ranging from highly specific to broadly cross-reactive. Umedais therefore also incorporated herein by reference for purposes offurther describing screening assays to identify MAbs that bindspecifically to PS, e.g., and do not bind to phosphatidylcholine.

Any of the 61 hybridomas generated by Umeda could potentially beemployed in the therapeutic agent-targeting agent constructs of thepresent invention. Examples are PSC8, PSF11, PSG3, PSD11, PSF10, PS1B,PS3D12, PS2C11; PS3A, PSF6, PSF7, PSB4, PS3H1; PS4A7 and PS1G3. Morepreferred are PS3A, PSF6, PSF7, PSB4 and PS3H1 as they bind only tophosphatidylserine and phosphatidylethanolamine. Preferred anti-PSantibodies are PS4A7 (IgM) and PS1G3 (IgG₃), as they are highly specificfor PS and exhibit no cross-reaction with other phospholipids. PS4A7recognizes the stereo-specific configuration of the serine residue in PS(FIG. 1 Umeda et al., 1989; incorporated herein by reference).

Igarashi et al. (1991; incorporated herein by reference) also reportedthe effective induction of anti-PS antibodies of the IgG isotype byintrasplenic immunization. Only a slight increase of the titer wasobserved when the antigen was again injected intravenously. A highfrequency of anti-PS MAbs of the IgG isotype was also observed even whenMAbs were produced 10 days after the intrasplenic injection of theantigen. These antibodies were also employed by Schuurmans Stekhoven etal. (1994).

The other significant anti-PS antibody production has been by Rote andcolleagues. Rote et al. (1993; incorporated herein by reference)particularly employed PS micelles in combination with Freund's completeadjuvant to generate specific anti-PS antibodies. Rote et al. (1993)also generated monoclonal antibodies that differentiate betweencardiolipin (CL) and PS. Rote et al. (1993) is therefore alsoincorporated herein by reference for purposes of further describingscreening assays to identify MAbs that bind specifically to PS bytesting against resting and thrombin-activated platelets using flowcytometry.

The 3SB9b antibody produced by Rote et al. (1993) reacted with only withPS, and is a preferred antibody for use in the therapeuticagent-targeting agent constructs of the present invention. BA3B5C4 mayalso be used as it reacts with both PS and CL. These antibodies are alsodescribed in Lin et al. (1995), Obringer et al. (1995) and Katsuragawaet al. (1997).

E3. Anti-Aminophospholipid Antibodies from Phagemid Libraries

Recombinant technology now allows the preparation of antibodies havingthe desired specificity from recombinant genes encoding a range ofantibodies (Van Dijk et al., 1989; incorporated herein by reference).Certain recombinant techniques involve the isolation of the antibodygenes by immunological screening of combinatorial immunoglobulin phageexpression libraries prepared from RNA isolated from the spleen of animmunized animal (Morrison et al., 1986; Winter and Milstein, 1991; eachincorporated herein by reference).

For such methods, combinatorial immunoglobulin phagemid libraries areprepared from RNA isolated from the spleen of the immunized animal, andphagemids expressing appropriate antibodies are selected by panningusing cells expressing the antigen and control cells. The advantages ofthis approach over conventional hybridoma techniques are thatapproximately 10⁴ times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination, which further increases the percentage of appropriateantibodies generated.

One method for the generation of a large repertoire of diverse antibodymolecules in bacteria utilizes the bacteriophage lambda as the vector(Huse et al., 1989; incorporated herein by reference). Production ofantibodies using the lambda vector involves the cloning of heavy andlight chain populations of DNA sequences into separate starting vectors.The vectors are subsequently combined randomly to form a single vectorthat directs the co-expression of heavy and light chains to formantibody fragments. The heavy and light chain DNA sequences are obtainedby amplification, preferably by PCR™ or a related amplificationtechnique, of mRNA isolated from spleen cells (or hybridomas thereof)from an animal that has been immunized with a selected antigen. Theheavy and light chain sequences are typically amplified using primersthat incorporate restriction sites into the ends of the amplified DNAsegment to facilitate cloning of the heavy and light chain segments intothe starting vectors.

Another method for the generation and screening of large libraries ofwholly or partially synthetic antibody combining sites, or paratopes,utilizes display vectors derived from filamentous phage such as M13, flor fd. These filamentous phage display vectors, referred to as“phagemids”, yield large libraries of monoclonal antibodies havingdiverse and novel immunospecificities. The technology uses a filamentousphage coat protein membrane anchor domain as a means for linkinggene-product and gene during the assembly stage of filamentous phagereplication, and has been used for the cloning and expression ofantibodies from combinatorial libraries (Kang et al., 1991; Barbas etal., 1991; each incorporated herein by reference).

This general technique for filamentous phage display is described inU.S. Pat. No. 5,658,727, incorporated herein by reference. In a mostgeneral sense, the method provides a system for the simultaneous cloningand screening of pre-selected ligand-binding specificities from antibodygene repertoires using a single vector system. Screening of isolatedmembers of the library for a pre-selected ligand-binding capacity allowsthe correlation of the binding capacity of an expressed antibodymolecule with a convenient means to isolate the gene that encodes themember from the library.

Linkage of expression and screening is accomplished by the combinationof targeting of a fusion polypeptide into the periplasm of a bacterialcell to allow assembly of a functional antibody, and the targeting of afusion polypeptide onto the coat of a filamentous phage particle duringphage assembly to allow for convenient screening of the library memberof interest. Periplasmic targeting is provided by the presence of asecretion signal domain in a fusion polypeptide. Targeting to a phageparticle is provided by the presence of a filamentous phage coat proteinmembrane anchor domain (i.e., a cpIII- or cpVIII-derived membrane anchordomain) in a fusion polypeptide.

The diversity of a filamentous phage-based combinatorial antibodylibrary can be increased by shuffling of the heavy and light chaingenes, by altering one or more of the complementarity determiningregions of the cloned heavy chain genes of the library, or byintroducing random mutations into the library by error-prone polymerasechain reactions. Additional methods for screening phagemid libraries aredescribed in U.S. Pat. No. 5,580,717; 5,427,908; 5,403,484; and5,223,409, each incorporated herein by reference.

Another method for the screening of large combinatorial antibodylibraries has been developed, utilizing expression of populations ofdiverse heavy and light chain sequences on the surface of a filamentousbacteriophage, such as M13, fl or fd (U.S. Pat. No. 5,698,426;incorporated herein by reference). Two populations of diverse heavy (Hc)and light (Lc) chain sequences are synthesized by polymerase chainreaction (PCR™). These populations are cloned into separate M13-basedvector containing elements necessary for expression. The heavy chainvector contains a gene VIII (gVIII) coat protein sequence so thattranslation of the heavy chain sequences produces gVIII-Hc fusionproteins. The populations of two vectors are randomly combined such thatonly the vector portions containing the Hc and Lc sequences are joinedinto a single circular vector.

The combined vector directs the co-expression of both Hc and Lcsequences for assembly of the two polypeptides and surface expression onM13 (U.S. Pat. No. 5,698,426; incorporated herein by reference). Thecombining step randomly brings together different Hc and Lc encodingsequences within two diverse populations into a single vector. Thevector sequences donated from each independent vector are necessary forproduction of viable phage. Also, since the pseudo gVIII sequences arecontained in only one of the two starting vectors, co-expression offunctional antibody fragments as Lc associated gVIII-Hc fusion proteinscannot be accomplished on the phage surface until the vector sequencesare linked in the single vector.

Surface expression of the antibody library is performed in an ambersuppressor strain. An amber stop codon between the Hc sequence and thegVIII sequence unlinks the two components in a non-suppressor strain.Isolating the phage produced from the non-suppressor strain andinfecting a suppressor strain will link the Hc sequences to the gVIIIsequence during expression. Culturing the suppressor strain afterinfection allows the coexpression on the surface of M13 of all antibodyspecies within the library as gVIII fusion proteins (gVIII-Fab fusionproteins). Alternatively, the DNA can be isolated from thenon-suppressor strain and then introduced into a suppressor strain toaccomplish the same effect.

The surface expression library is screened for specific Fab fragmentsthat bind preselected molecules by standard affinity isolationprocedures. Such methods include, for example, panning (Parmley andSmith, 1988; incorporated herein by reference), affinity chromatographyand solid phase blotting procedures. Panning is preferred, because hightiters of phage can be screened easily, quickly and in small volumes.Furthermore, this procedure can select minor Fab fragments specieswithin the population, which otherwise would have been undetectable, andamplified to substantially homogenous populations. The selected Fabfragments can be characterized by sequencing the nucleic acids encodingthe polypeptides after amplification of the phage population.

Another method for producing diverse libraries of antibodies andscreening for desirable binding specificities is described in U.S. Pat.No. 5,667,988 and U.S. Pat. No. 5,759,817, each incorporated herein byreference. The method involves the preparation of libraries ofheterodimeric immunoglobulin molecules in the form of phagemid librariesusing degenerate oligonucleotides and primer extension reactions toincorporate the degeneracies into the CDR regions of the immunoglobulinvariable heavy and light chain variable domains, and display of themutagenized polypeptides on the surface of the phagemid. Thereafter, thedisplay protein is screened for the ability to bind to a preselectedantigen.

The method for producing a heterodimeric immunoglobulin moleculegenerally involves (1) introducing a heavy or light chain Vregion-coding gene of interest into the phagemid display vector; (2)introducing a randomized binding site into the phagemid display proteinvector by primer extension with an oligonucleotide containing regions ofhomology to a CDR of the antibody V region gene and containing regionsof degeneracy for producing randomized coding sequences to form a largepopulation of display vectors each capable of expressing differentputative binding sites displayed on a phagemid surface display protein;(3) expressing the display protein and binding site on the surface of afilamentous phage particle; and (4) isolating (screening) thesurface-expressed phage particle using affinity techniques such aspanning of phage particles against a preselected antigen, therebyisolating one or more species of phagemid containing a display proteincontaining a binding site that binds a preselected antigen.

A further variation of this method for producing diverse libraries ofantibodies and screening for desirable binding specificities isdescribed in U.S. Pat. No. 5,702,892, incorporated herein by reference.In this method, only heavy chain sequences are employed, the heavy chainsequences are randomized at all nucleotide positions which encode eitherthe CDRI or CDRIII hypervariable region, and the genetic variability inthe CDRs is generated independent of any biological process.

In the method, two libraries are engineered to genetically shuffleoligonucleotide motifs within the framework of the heavy chain genestructure. Through random mutation of either CDRI or CDRIII, thehypervariable regions of the heavy chain gene were reconstructed toresult in a collection of highly diverse sequences. The heavy chainproteins encoded by the collection of mutated gene sequences possessedthe potential to have all of the binding characteristics of animmunoglobulin while requiring only one of the two immunoglobulinchains.

Specifically, the method is practiced in the absence of theimmunoglobulin light chain protein. A library of phage displayingmodified heavy chain proteins is incubated with an immobilized ligand toselect clones encoding recombinant proteins that specifically bind theimmobilized ligand. The bound phage are then dissociated from theimmobilized ligand and amplified by growth in bacterial host cells.Individual viral plaques, each expressing a different recombinantprotein, are expanded, and individual clones can then be assayed forbinding activity.

E4. Anti-Aminophospholipid Antibodies from Human Patients

Antibodies against aminophospholipids, particularly phosphatidylserineand phosphatidylethanolamine, occur in the human population, where theyare correlated with certain disease states. Anti-aminophospholipidantibodies are part of the heterogeneous anti-phospholipid antibodies(aPL), observed to have families of different specificities and classes.Primary anti-phospholipid syndrome (APS) has even been separated fromother forms of autoimmune disease associated with anti-phospholipidantibody production.

Anti-PS antibodies are particularly associated with recurrent pregnancyloss (Rote et al., 1995; Rote, 1996; Vogt et al., 1996; Vogt et al.,1997; incorporated herein by reference) and with the autoimmune disease,systemic lupus erythematosus (SLE or “lupus”) (Branch et al., 1987;incorporated herein by reference). Anti-PE antibodies have also beenreported in human patients, particularly those with autoimmune diseases(Staub et al., 1989). Branch et al. (1987) reported that 80% of patientswith lupus anticoagulant (LA or LAC) had autoantibodies that recognizedPE; with Drouvalakis and Buchanan (1998) increasing this number to 95%PE-positives from autoimmune LAC sera.

Anti-phospholipid antibodies are not to be confused withanti-endothelial cell antibodies (AECA), although they can be found inthe same patient. The existence of AECA has been documented in a varietyof clinical settings associated with vasculitis, such as systemicsclerosis (SS). To study AECA, antibodies are obtained from patientsthat do not have anti-phospholipid antibodies (aPL-negative sera).

The pathogenic role of AECA remains unclear, although Bordron et al.(1998) very recently suggested that AECA may initiate apoptosis inendothelial cells, which would be followed by PS transfer to the outerface of the membrane. They proposed that this would account for thesubsequent generation of the anti-phospholipid antibodies that aresometimes seen in conjunction with AECA in patients with skin lesions orconnective tissue disease (Bordron et al., 1998). However, although AECAbinding to an apoptosis-inducing antigen was postulated, these studiesdid not lead to the further characterization of AECA, still said torepresent an extremely heterogeneous family of antibodies reacting withdifferent (non-lipid) structures on endothelial cells (Bordron et al.,1998).

Anti-phosphatidylserine antibodies are closely associated with pregnancyloss, pregnancy-induced hypertension and intrauterine growthretardation. A phosphatidylserine-dependent antigen has been shown to beexpressed on the surface of a choriocarcinoma model (BeWo) ofdifferentiating cytotrophoblastic cells, indicating that it should beaccessible in vivo to circulating anti-phosphatidylserine antibodies(Rote et al., 1995). Indeed, Vogt et al. (1996) showed that themonoclonal antibody 3SB9b, which reacts with phosphatidylserine but notcardiolipin, induced a significant reduction in both fetal and placentalweights in a mouse model for the anti-phospholipid antibody syndrome.

These authors developed a model for explaining miscarriages associatedwith anti-phospholipid antibodies: anti-phosphatidylserine antibodyreveals sites for prothrombin binding on the surface of the trophoblast,most likely by removing Annexin V (Vogt et al., 1997). Trophoblastdifferentiation is associated with externalization of phosphatidylserinefrom the inner to the outer surface of the plasma membrane. Normally,externalization of phosphatidylserine is concurrent with binding ofAnnexin V, which prevents the phosphatidylserine-rich surface fromacting as a site for activation of coagulation. Thus, whenanti-phospholipid antibodies are present, they prevent Annexin V bindingand lead to a procoagulant state (Vogt et al., 1997).

Anti-PE antibodies are frequently associated with lupus anticoagulants(LAC sera). The role of PE and anti-PE in LAC is extremely complex, see,e.g., Smirnov et al. (1995; incorporated herein by reference), wherevarious hypotheses are set forth. Smirnov et al. (1995) report that, inthe presence of activated protein C and PE, LAC plasma clots faster thannormal plasma. Rauch et al. (1986) characterize LAC anti-phospholipidantibodies as prolonging the clotting time in in vitro coagulationassays.

Vlachoyiannopoulos et al. (1993; incorporated herein by reference)tested SLE and APS sera by ELISA for antibodies tophosphatidylethanolamine and cardiolipin, as compared to healthy blooddonors. Both SLE and APS patients were reported to present a highertiter of IgM anti-PE antibodies than normal subjects, while the IgG andIgA anti-PE reactivity reportedly did not differ. It was suggested thatIgA and IgG anti-PE antibodies may occur in low titers as naturalautoantibodies in normal subjects (Vlachoyiannopoulos et al., 1993;incorporated herein by reference).

Rauch et al. (1986; incorporated herein by reference) producedhybridomas by fusing lymphocytes from 13 systemic lupus erythematosuspatients with a lymphoblastoid line. They demonstrated that theautoantibodies that prolonged clotting time bound to hexagonal phasephospholipids, including natural and synthetic forms ofphosphatidylethanolamine (Rauch et al., 1986; incorporated herein byreference). In contrast, lamellar phospholipids, such asphosphatidylcholine and synthetic lamellar forms ofphosphatidylethanolamine, had no effect on the anticoagulant activity(Rauch et al., 1986).

Rauch and Janoff (1990; incorporated herein by reference) went on toshow that immunization of mice with phosphatidylethanolamine in thehexagonal II phase, but not in the bilayer phase, resulted in theinduction of anti-phospholipid antibodies. These antibodies werestrongly reactive with phosphatidylethanolamine and had functional lupusanticoagulant activity characteristic of autoantibodies from patientswith autoimmune disease (Rauch and Janoff, 1990).

The hexagonal II phase form of aminophospholipids should thus beadvantageously used to generate antibodies for use in the presentinvention. Indeed, Trudell reported that antibodies raised against TFA-(trifluoroacetyl-) protein adducts bind to TFA-phosphatidylethanolaminein hexagonal phase phospholipid micelles, but not in lamellar liposomes(Trudell et al., 1991a; incorporated herein by reference). The authorssuggested that TFA-phosphatidylethanolamine adducts that reside innon-lamellar domains on the hepatocyte surface could be recognitionsites for anti-TFA-adduct antibodies and potentially participate inimmune-mediated halothane hepatotoxicity (Trudell et al., 1991a). It waslater shown that these same antibodies cross-react withTFA-dioleoylphosphatidylethanolamine when this adduct is incorporatedinto the surface of hepatocytes (Trudell et al., 1991b; incorporatedherein by reference), thus supporting this hypothesis.

Berard further explained the hexagonal II phase form ofaminophospholipids, such as PE (Berard et al., 1993; incorporated hereinby reference). In bilayers, phospholipids generally adopt a gelstructure, crystalline lattice or lamellar phase (Berard et al., 1993).However, depending on the cholesterol content, protein and ionicenvironments, phospholipids can easily change phases, adopting ahexagonal II phase (Berard et al., 1993; incorporated herein byreference). It is this hexagonal II phase of aminophospholipids that isbelieved to be immunogenic, as initial proposed for autoantibodygeneration in disease situations (Berard et al., 1993; incorporatedherein by reference).

Qamar et al. (1990; incorporated herein by reference) have developed avariation on the hexagonal aminophospholipid recognition theme. Usingphosphatidylethanolamine as a model, these authors reported that anti-PEantibodies from aPL-positive SLE sera do not bind to PE, but in fact aredirected to lysophosphatidylethanolamine (1PE), a natural PE degradationproduct and a likely contaminant of most PE preparations (Qamar et al.,1990; incorporated herein by reference).

Other recent data indicate that most anti-phospholipid antibodiesrecognize phospholipid in the context of nearby proteins (Rote, 1996;Chamley et al., 1991). In plasma membranes, the majority of thephospholipid appears to be naturally in non-antigenic bilaminar form(Rote, 1996). Accessory molecules may help facilitate the transition tohexagonal antigenic forms and stabilize their expression (Galli et al.,1993). For example, naturally occurring anti-phospholipid antibodieswere first reported to recognize complexes of cardiolipin orphosphatidylserine with β₂-glycoprotein I (β₂-GPI or apolipoprotein H,apoH) (Galli et al., 1990; 1993). β₂-GPI is believed to stabilizephospholipids in antigenic conformations that do not exist in purephospholipids (McNeil et al., 1990; U.S. Pat. No. 5,344,758; Chamley etal., 1991; Matsuura et al., 1994). Prothrombin has also been implicatedin the phospholipid stabilization process (Bevers et al., 1991).

Phospholipid-binding plasma proteins are also generally necessary forantibody recognition of the electrically neutral or zwitterionicphospholipid, phosphatidylethanolamine. Sugi and McIntyre (1995;incorporated herein by reference) identified two prominent PE-bindingplasma proteins as high molecular weight kininogen (HMWK or HK) and lowmolecular weight kininogen (LMWK or LK). Anti-PE antibodies frompatients with SLE and/or recurrent spontaneous abortions were shown notto recognize PE, HMWK or LMWK when they were presented independently assole antigens on ELISA plates (Sugi and McIntyre, 1995). Otheranti-PE-positive sera that did not react with PE-HMWK or PE-LMWK weresuggested to recognize factor XI or prekallikrein, which normally bindto HMWK (Sugi and McIntyre, 1995; incorporated herein by reference).

The validity of these results was confirmed by showing that intact HMWKbinds to various phospholipids, such as cardiolipin, phosphatidylserine,phosphatidylcholine and phosphatidylethanolamine; but that anti-PEantibodies recognize only a kininogen-PE complex, and do not recognizekininogens presented with other phospholipid substrates (Sugi andMcIntyre, 1996a; incorporated herein by reference). This indicates thatPE induces unique antigenic conformational changes in the kininogensthat are not induced when the kininogens bind to other phospholipids(Sugi and McIntyre, 1996a).

It has further been suggested that kininogens can bind to platelets byvirtue of exposed PE in the platelet membrane (Sugi and McIntyre, 1996b;incorporated herein by reference). Exogenously added kininogen-dependentanti-PE was shown to increase thrombin-induced platelet aggregation invitro, but not to alter ADP-induced aggregation (Sugi and McIntyre,1996b; incorporated herein by reference). In contrast, kininogenindependent anti-PE, which recognized PE per se, was reported notaugment thrombin-induced platelet aggregation. It was thus proposed thatkininogen dependent anti-PE may disrupt the normal anti-thromboticeffects of kininogen (Sugi and McIntyre, 1996b; incorporated herein byreference).

Anti-aminophospholipid antibodies from human patients are therefore amixture of antibodies that generally recognize aminophospholipidsstabilized by protein interactions (Rote, 1996). The antibodies may bindto stabilized phospholipid epitopes, or may bind to an epitope formedfrom the interaction of the phospholipid and amino acids on thestabilizing protein (Rote, 1996). Either way, such antibodies clearlyrecognize aminophospholipids in natural membranes in the human body,probably associated with plasma proteins (McNeil et al., 1990; Bevers etal., 1991). These antibodies would thus be appropriate as startingmaterials for generating an antibody for use in the therapeuticagent-targeting agent constructs of the present invention.

To prepare an anti-aminophospholipid antibody from a human patient, onewould simply obtain human lymphocytes from an individual havinganti-aminophospholipid antibodies, for example from human peripheralblood, spleen, lymph nodes, tonsils or the like, utilizing techniquesthat are well known to those of skill in the art. The use of peripheralblood lymphocytes will often be preferred.

Human monoclonal antibodies may be obtained from the human lymphocytesproducing the desired anti-aminophospholipid antibodies by immortalizingthe human lymphocytes, generally in the same manner as described abovefor generating any monoclonal antibody. The reactivities of theantibodies in the culture supernatants are generally first checked,employing one or more selected aminophospholipid antigen(s), and thelymphocytes that exhibit high reactivity are grown. The resultinglymphocytes are then fused with a parent line of human or mouse origin,and further selection gives the optimal clones.

The recovery of monoclonal antibodies from the immortalized cells may beachieved by any method generally employed in the production ofmonoclonal antibodies. For instance, the desired monoclonal antibody maybe obtained by cloning the immortalized lymphocyte by the limitingdilution method or the like, selecting the cell producing the desiredantibody, growing the selected cells in a medium or the abdominal cavityof an animal, and recovering the desired monoclonal antibody from theculture supernatant or ascites.

Such techniques have been used, for example, to isolate human monoclonalantibodies to Pseudomonas aeruginosa epitopes (U.S. Pat. Nos. 5,196,337and 5,252,480, each incorporated herein by reference);polyribosylribitol phosphate capsular polysaccharides (U.S. Pat. No.4,954,449, incorporated herein by reference); the Rh(D) antigen (U.S.Pat. No. 5,665,356, incorporated herein by reference); and viruses, suchas human immunodeficiency virus, respiratory syncytial virus, herpessimplex virus, varicella zoster virus and cytomegalovirus (U.S. Pat.Nos. 5,652,138; 5,762,905; and 4,950,595, each incorporated herein byreference).

The applicability of the foregoing techniques to the generation of humananti-aminophospholipid antibodies is clear. Rauch et al. (1986;incorporated herein by reference) generally used such methods to producehybridomas by fusing lymphocytes from SLE patients with a lymphoblastoidline. This produced human antibodies that bound to hexagonal phasephospholipids, including natural and synthetic forms ofphosphatidylethanolamine (Rauch et al., 1986; incorporated herein byreference).

Additionally, the methods described in U.S. Pat. No. 5,648,077(incorporated herein by reference) can be used to form a trioma or aquadroma that produces a human antibody against a selectedaminophospholipid. In a general sense, a hybridoma cell line comprisinga parent rodent immortalizing cell, such as a murine myeloma cell, e.g.SP-2, is fused to a human partner cell, resulting in an immortalizingxenogeneic hybridoma cell. This xenogeneic hybridoma cell is fused to acell capable of producing an anti-aminophospholipid human antibody,resulting in a trioma cell line capable of generating human antibodyeffective against such antigen in a human. Alternately, when greaterstability is desired, a trioma cell line which preferably no longer hasthe capability of producing its own antibody is made, and this trioma isthen fused with a further cell capable of producing an antibody usefulagainst the aminophospholipid antigen to obtain a still more stablehybridoma (quadroma) that produces antibody against the antigen.

E5. Anti-Aminophospholipid Antibodies from Human Lymphocytes

In vitro immunization, or antigen stimulation, may also be used togenerate a human anti-aminophospholipid antibody. Such techniques can beused to stimulate peripheral blood lymphocytes from bothanti-aminophospholipid antibody-producing human patients, and also fromnormal, healthy subjects. Indeed, Vlachoyiannopoulos et al. (1993;incorporated herein by reference) reported that low titeranti-aminophospholipid antibodies occur in normal subjects. Even if thiswere not the case, anti-aminophospholipid antibodies can be preparedfrom healthy human subjects, simply by stimulating antibody-producingcells with aminophospholipids in vitro.

Such “in vitro immunization” involves antigen-specific activation ofnon-immunized B lymphocytes, generally within a mixed population oflymphocytes (mixed lymphocyte cultures, MLC). In vitro immunizations mayalso be supported by B cell growth and differentiation factors andlymphokines. The antibodies produced by these methods are often IgMantibodies (Borrebaeck et al., 1986; incorporated herein by reference).

Another method has been described (U.S. Pat. No. 5,681,729, incorporatedherein by reference) wherein human lymphocytes that mainly produce IgG(or IgA) antibodies can be obtained. The method involves, in a generalsense, transplanting human lymphocytes to an immunodeficient animal sothat the human lymphocytes “take” in the animal body; immunizing theanimal with a desired antigen, so as to generate human lymphocytesproducing an antibody specific to the antigen; and recovering the humanlymphocytes producing the antibody from the animal. The humanlymphocytes thus produced can be used to produce a monoclonal antibodyby immortalizing the human lymphocytes producing the antibody, cloningthe obtained immortalized human-originated lymphocytes producing theantibody, and recovering a monoclonal antibody specific to the desiredantigen from the cloned immortalized human-originated lymphocytes.

The immunodeficient animals that may be employed in this technique arethose that do not exhibit rejection when human lymphocytes aretransplanted to the animals. Such animals may be artificially preparedby physical, chemical or biological treatments. Any immunodeficientanimal may be employed. The human lymphocytes may be obtained from humanperipheral blood, spleen, lymph nodes, tonsils or the like.

The “taking” of the transplanted human lymphocytes in the animals can beattained by merely administering the human lymphocytes to the animals.The administration route is not restricted and may be, for example,subcutaneous, intravenous or intraperitoneal. The dose of the humanlymphocytes is not restricted, and can usually be 10⁶ to 10⁸ lymphocytesper animal. The immunodeficient animal is then immunized with thedesired aminophospholipid antigen.

After the immunization, human lymphocytes are recovered from the blood,spleen, lymph nodes or other lymphatic tissues by any conventionalmethod. For example, mononuclear cells can be separated by theFicoll-Hypaque (specific gravity: 1.077) centrifugation method, and themonocytes removed by the plastic dish adsorption method. Thecontaminating cells originating from the immunodeficient animal may beremoved by using an antiserum specific to the animal cells. Theantiserum may be obtained by, for example, immunizing a second, distinctanimal with the spleen cells of the immunodeficient animal, andrecovering serum from the distinct immunized animal. The treatment withthe antiserum may be carried out at any stage. The human lymphocytes mayalso be recovered by an immunological method employing a humanimmunoglobulin expressed on the cell surface as a marker.

By these methods, human lymphocytes mainly producing IgG and IgAantibodies specific to one or more selected aminophospholipid(s) can beobtained. Monoclonal antibodies are then obtained from the humanlymphocytes by immortalization, selection, cell growth and antibodyproduction.

E6. Transgenic Mice Containing Human Antibody Libraries

Recombinant technology is now available for the preparation ofantibodies. In addition to the combinatorial immunoglobulin phageexpression libraries disclosed above, another molecular cloning approachis to prepare antibodies from transgenic mice containing human antibodylibraries. Such techniques are described in U.S. Pat. No. 5,545,807,incorporated herein by reference.

In a most general sense, these methods involve the production of atransgenic animal that has inserted into its germline genetic materialthat encodes for at least part of an immunoglobulin of human origin orthat can rearrange to encode a repertoire of immunoglobulins. Theinserted genetic material may be produced from a human source, or may beproduced synthetically. The material may code for at least part of aknown immunoglobulin or may be modified to code for at least part of analtered immunoglobulin.

The inserted genetic material is expressed in the transgenic animal,resulting in production of an immunoglobulin derived at least in partfrom the inserted human immunoglobulin genetic material. It is found thegenetic material is rearranged in the transgenic animal, so that arepertoire of immunoglobulins with part or parts derived from insertedgenetic material may be produced, even if the inserted genetic materialis incorporated in the germline in the wrong position or with the wronggeometry.

The inserted genetic material may be in the form of DNA cloned intoprokaryotic vectors such as plasmids and/or cosmids. Larger DNAfragments are inserted using yeast artificial chromosome vectors (Burkeet al., 1987; incorporated herein by reference), or by introduction ofchromosome fragments (Richer and Lo, 1989; incorporated herein byreference). The inserted genetic material may be introduced to the hostin conventional manner, for example by injection or other proceduresinto fertilized eggs or embryonic stem cells.

In preferred aspects, a host animal that initially does not carrygenetic material encoding immunoglobulin constant regions is utilized,so that the resulting transgenic animal will use only the inserted humangenetic material when producing immunoglobulins. This can be achievedeither by using a naturally occurring mutant host lacking the relevantgenetic material, or by artificially making mutants e.g., in cell linesultimately to create a host from which the relevant genetic material hasbeen removed.

Where the host animal carries genetic material encoding immunoglobulinconstant regions, the transgenic animal will carry the naturallyoccurring genetic material and the inserted genetic material and willproduce immunoglobulins derived from the naturally occurring geneticmaterial, the inserted genetic material, and mixtures of both types ofgenetic material. In this case the desired immunoglobulin can beobtained by screening hybridomas derived from the transgenic animal,e.g., by exploiting the phenomenon of allelic exclusion of antibody geneexpression or differential chromosome loss.

Once a suitable transgenic animal has been prepared, the animal issimply immunized with the desired immunogen. Depending on the nature ofthe inserted material, the animal may produce a chimeric immunoglobulin,e.g. of mixed mouse/human origin, where the genetic material of foreignorigin encodes only part of the immunoglobulin; or the animal mayproduce an entirely foreign immunoglobulin, e.g. of wholly human origin,where the genetic material of foreign origin encodes an entireimmunoglobulin.

Polyclonal antisera may be produced from the transgenic animal followingimmunization. Immunoglobulin-producing cells may be removed from theanimal to produce the immunoglobulin of interest. Preferably, monoclonalantibodies are produced from the transgenic animal, e.g., by fusingspleen cells from the animal with myeloma cells and screening theresulting hybridomas to select those producing the desired antibody.Suitable techniques for such processes are described herein.

In an alternative approach, the genetic material may be incorporated inthe animal in such a way that the desired antibody is produced in bodyfluids such as serum or external secretions of the animal, such as milk,colostrum or saliva. For example, by inserting in vitro genetic materialencoding for at least part of a human immunoglobulin into a gene of amammal coding for a milk protein and then introducing the gene to afertilized egg of the mammal, e.g., by injection, the egg may developinto an adult female mammal producing milk containing immunoglobulinderived at least in part from the inserted human immunoglobulin geneticmaterial. The desired antibody can then be harvested from the milk.Suitable techniques for carrying out such processes are known to thoseskilled in the art.

The foregoing transgenic animals are usually employed to produce humanantibodies of a single isotype, more specifically an isotype that isessential for B cell maturation, such as IgM and possibly IgD. Anotherpreferred method for producing human anti-aminophospholipid antibodiesis to use the technology described in U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,770,429; eachincorporated by reference, wherein transgenic animals are described thatare capable of switching from an isotype needed for B cell developmentto other isotypes.

In the development of a B lymphocyte, the cell initially produces IgMwith a binding specificity determined by the productively rearrangedV_(H) and V_(L) regions. Subsequently, each B cell and its progeny cellssynthesize antibodies with the same L and H chain V regions, but theymay switch the isotype of the H chain. The use of mu or delta constantregions is largely determined by alternate splicing, permitting IgM andIgD to be coexpressed in a single cell. The other heavy chain isotypes(gamma, alpha, and epsilon) are only expressed natively after a generearrangement event deletes the C mu and C delta exons. This generearrangement process, termed isotype switching, typically occurs byrecombination between so called switch segments located immediatelyupstream of each heavy chain gene (except delta). The individual switchsegments are between 2 and 10 kb in length, and consist primarily ofshort repeated sequences.

For these reasons, it is preferable that transgenes incorporatetranscriptional regulatory sequences within about 1–2 kb upstream ofeach switch region that is to be utilized for isotype switching. Thesetranscriptional regulatory sequences preferably include a promoter andan enhancer element, and more preferably include the 5′ flanking (i.e.,upstream) region that is naturally associated (i.e., occurs in germlineconfiguration) with a switch region. Although a 5′ flanking sequencefrom one switch region can be operably linked to a different switchregion for transgene construction, in some embodiments it is preferredthat each switch region incorporated in the transgene construct have the5′ flanking region that occurs immediately upstream in the naturallyoccurring germline configuration. Sequence information relating toimmunoglobulin switch region sequences is known (Mills et al., 1990;Sideras et al., 1989; each incorporated herein by reference).

In the method described in U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,661,016; and 5,770,429, the human immunoglobulintransgenes contained within the transgenic animal function correctlythroughout the pathway of B-cell development, leading to isotypeswitching. Accordingly, in this method, these transgenes are constructedso as to produce isotype switching and one or more of the following: (1)high level and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

An important requirement for transgene function is the generation of aprimary antibody repertoire that is diverse enough to trigger asecondary immune response for a wide range of antigens. The rearrangedheavy chain gene consists of a signal peptide exon, a variable regionexon and a tandem array of multi-domain constant region regions, each ofwhich is encoded by several exons. Each of the constant region genesencode the constant portion of a different class of immunoglobulins.During B-cell development, V region proximal constant regions aredeleted leading to the expression of new heavy chain classes. For eachheavy chain class, alternative patterns of RNA splicing give rise toboth transmembrane and secreted immunoglobulins.

The human heavy chain locus consists of approximately 200 V genesegments spanning 2 Mb, approximately 30 D gene segments spanning about40 kb, six J segments clustered within a 3 kb span, and nine constantregion gene segments spread out over approximately 300 kb. The entirelocus spans approximately 2.5 Mb of the distal portion of the long armof chromosome 14. Heavy chain transgene fragments containing members ofall six of the known V_(H) families, the D and J gene segments, as wellas the mu, delta, gamma 3, gamma 1 and alpha 1 constant regions areknown (Berman et al., 1988; incorporated herein by reference). Genomicfragments containing all of the necessary gene segments and regulatorysequences from a human light chain locus is similarly constructed.

The expression of successfully rearranged immunoglobulin heavy and lighttransgenes usually has a dominant effect by suppressing therearrangement of the endogenous immunoglobulin genes in the transgenicnonhuman animal. However, in certain embodiments, it is desirable toeffect complete inactivation of the endogenous Ig loci so that hybridimmunoglobulin chains comprising a human variable region and a non-human(e.g., murine) constant region cannot be formed, for example bytrans-switching between the transgene and endogenous Ig sequences. Usingembryonic stem cell technology and homologous recombination, theendogenous immunoglobulin repertoire can be readily eliminated. Inaddition, suppression of endogenous Ig genes may be accomplished using avariety of techniques, such as antisense technology.

In other aspects of the invention, it may be desirable to produce atrans-switched immunoglobulin. Antibodies comprising such chimerictrans-switched immunoglobulins can be used for a variety of applicationswhere it is desirable to have a non-human (e.g., murine) constantregion, e.g., for retention of effector functions in the host. Thepresence of a murine constant region can afford advantages over a humanconstant region, for example, to provide murine effector functions(e.g., ADCC, murine complement fixation) so that such a chimericantibody may be tested in a mouse disease model. Subsequent to theanimal testing, the human variable region encoding sequence may beisolated, e.g., by PCR amplification or cDNA cloning from the source(hybridoma clone), and spliced to a sequence encoding a desired humanconstant region to encode a human sequence antibody more suitable forhuman therapeutic use.

E7. Humanized Anti-Aminophospholipid Antibodies

Human antibodies generally have at least three potential advantages foruse in human therapy. First, because the effector portion is human, itmay interact better with the other parts of the human immune system,e.g., to destroy target cells more efficiently by complement-dependentcytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).Second, the human immune system should not recognize the antibody asforeign. Third, the half-life in the human circulation will be similarto naturally occurring human antibodies, allowing smaller and lessfrequent doses to be given.

Various methods for preparing human anti-aminophospholipids are providedherein. In addition to human antibodies, “humanized” antibodies havemany advantages. “Humanized” antibodies are generally chimeric or mutantmonoclonal antibodies from mouse, rat, hamster, rabbit or other species,bearing human constant and/or variable region domains or specificchanges. Techniques for generating a so-called “humanized”anti-aminophospholipid antibody are well known to those of skill in theart.

Humanized antibodies also share the foregoing advantages. First, theeffector portion is still human. Second, the human immune system shouldnot recognize the framework or constant region as foreign, and thereforethe antibody response against such an injected antibody should be lessthan against a totally foreign mouse antibody. Third, injected humanizedantibodies, as opposed to injected mouse antibodies, will presumablyhave a half-life more similar to naturally occurring human antibodies,also allowing smaller and less frequent doses.

A number of methods have been described to produce humanized antibodies.Controlled rearrangement of antibody domains joined through proteindisulfide bonds to form new, artificial protein molecules or “chimeric”antibodies can be utilized (Konieczny et al., 1981; incorporated hereinby reference). Recombinant DNA technology can also be used to constructgene fusions between DNA sequences encoding mouse antibody variablelight and heavy chain domains and human antibody light and heavy chainconstant domains (Morrison et al., 1984; incorporated herein byreference).

DNA sequences encoding the antigen binding portions or complementaritydetermining regions (CDR's) of murine monoclonal antibodies can begrafted by molecular means into the DNA sequences encoding theframeworks of human antibody heavy and light chains (Jones et al., 1986;Riechmann et al., 1988; each incorporated herein by reference). Theexpressed recombinant products are called “reshaped” or humanizedantibodies, and comprise the framework of a human antibody light orheavy chain and the antigen recognition portions, CDR's, of a murinemonoclonal antibody.

Another method for producing humanized antibodies is described in U.S.Pat. No. 5,639,641, incorporated herein by reference. The methodprovides, via resurfacing, humanized rodent antibodies that haveimproved therapeutic efficacy due to the presentation of a human surfacein the variable region. In the method: (1) position alignments of a poolof antibody heavy and light chain variable regions is generated to givea set of heavy and light chain variable region framework surface exposedpositions, wherein the alignment positions for all variable regions areat least about 98% identical; (2) a set of heavy and light chainvariable region framework surface exposed amino acid residues is definedfor a rodent antibody (or fragment thereof); (3) a set of heavy andlight chain variable region framework surface exposed amino acidresidues that is most closely identical to the set of rodent surfaceexposed amino acid residues is identified; (4) the set of heavy andlight chain variable region framework surface exposed amino acidresidues defined in step (2) is substituted with the set of heavy andlight chain variable region framework surface exposed amino acidresidues identified in step (3), except for those amino acid residuesthat are within 5 Å of any atom of any residue of the complementaritydetermining regions of the rodent antibody; and (5) the humanized rodentantibody having binding specificity is produced.

A similar method for the production of humanized antibodies is describedin U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101, eachincorporated herein by reference. These methods involve producinghumanized immunoglobulins having one or more complementarity determiningregions (CDR's) and possible additional amino acids from a donorimmunoglobulin and a framework region from an accepting humanimmunoglobulin. Each humanized immunoglobulin chain usually comprises,in addition to the CDR's, amino acids from the donor immunoglobulinframework that are capable of interacting with the CDR's to effectbinding affinity, such as one or more amino acids that are immediatelyadjacent to a CDR in the donor immunoglobulin or those within about 3 Åas predicted by molecular modeling. The heavy and light chains may eachbe designed by using any one, any combination, or all of the variousposition criteria described in U.S. Pat. Nos. 5,693,762; 5,693,761;5,585,089; and 5,530,101, each incorporated herein by reference. Whencombined into an intact antibody, the humanized immunoglobulins aresubstantially non-immunogenic in humans and retain substantially thesame affinity as the donor immunoglobulin to the original antigen.

An additional method for producing humanized antibodies is described inU.S. Pat. Nos. 5,565,332 and 5,733,743, each incorporated herein byreference. This method combines the concept of humanizing antibodieswith the phagemid libraries also described in detail herein. In ageneral sense, the method utilizes sequences from the antigen bindingsite of an antibody or population of antibodies directed against anantigen of interest. Thus for a single rodent antibody, sequencescomprising part of the antigen binding site of the antibody may becombined with diverse repertoires of sequences of human antibodies thatcan, in combination, create a complete antigen binding site.

The antigen binding sites created by this process differ from thosecreated by CDR grafting, in that only the portion of sequence of theoriginal rodent antibody is likely to make contacts with antigen in asimilar manner. The selected human sequences are likely to differ insequence and make alternative contacts with the antigen from those ofthe original binding site. However, the constraints imposed by bindingof the portion of original sequence to antigen and the shapes of theantigen and its antigen binding sites, are likely to drive the newcontacts of the human sequences to the same region or epitope of theantigen. This process has therefore been termed “epitope imprintedselection” (EIS).

Starting with an animal antibody, one process results in the selectionof antibodies that are partly human antibodies. Such antibodies may besufficiently similar in sequence to human antibodies to be used directlyin therapy or after alteration of a few key residues. Sequencedifferences between the rodent component of the selected antibody withhuman sequences could be minimized by replacing those residues thatdiffer with the residues of human sequences, for example, by sitedirected mutagenesis of individual residues, or by CDR grafting ofentire loops. However, antibodies with entirely human sequences can alsobe created. EIS therefore offers a method for making partly human orentirely human antibodies that bind to the same epitope as animal orpartly human antibodies respectively. In EIS, repertoires of antibodyfragments can be displayed on the surface of filamentous phase and thegenes encoding fragments with antigen binding activities selected bybinding of the phage to antigen.

Additional methods for humanizing antibodies contemplated for use in thepresent invention are described in U.S. Pat. Nos. 5,750,078; 5,502,167;5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and4,816,567, each incorporated herein by reference.

E8. Mutagenesis by PCR

Site-specific mutagenesis is a technique useful in the preparation ofindividual antibodies through specific mutagenesis of the underlyingDNA. The technique further provides a ready ability to prepare and testsequence variants, incorporating one or more of the foregoingconsiderations, whether humanizing or not, by introducing one or morenucleotide sequence changes into the DNA.

Although many methods are suitable for use in mutagenesis, the use ofthe polymerase chain reaction (PCR™) is generally now preferred. Thistechnology offers a quick and efficient method for introducing desiredmutations into a given DNA sequence. The following text particularlydescribes the use of PCR™ to introduce point mutations into a sequence,as may be used to change the amino acid encoded by the given sequence.Adaptations of this method are also suitable for introducing restrictionenzyme sites into a DNA molecule.

In this method, synthetic oligonucleotides are designed to incorporate apoint mutation at one end of an amplified segment. Following PCR™, theamplified fragments are blunt-ended by treating with Klenow fragments,and the blunt-ended fragments are then ligated and subcloned into avector to facilitate sequence analysis.

To prepare the template DNA that one desires to mutagenize, the DNA issubcloned into a high copy number vector, such as pUC19, usingrestriction sites flanking the area to be mutated. Template DNA is thenprepared using a plasmid miniprep. Appropriate oligonucleotide primersthat are based upon the parent sequence, but which contain the desiredpoint mutation and which are flanked at the 5′ end by a restrictionenzyme site, are synthesized using an automated synthesizer. It isgenerally required that the primer be homologous to the template DNA forabout 15 bases or so. Primers may be purified by denaturingpolyacrylamide gel electrophoresis, although this is not absolutelynecessary for use in PCR™. The 5′ end of the oligonucleotides shouldthen be phosphorylated.

The template DNA should be amplified by PCR™, using the oligonucleotideprimers that contain the desired point mutations. The concentration ofMgCl₂ in the amplification buffer will generally be about 15 mM.Generally about 20–25 cycles of PCR™ should be carried out as follows:denaturation, 35 sec. at 95° C.; hybridization, 2 min. at 50° C.; andextension, 2 min. at 72° C. The PCR™ will generally include a last cycleextension of about 10 min. at 72° C. After the final extension step,about 5 units of Klenow fragments should be added to the reactionmixture and incubated for a further 15 min. at about 30° C. Theexonuclease activity of the Klenow fragments is required to make theends flush and suitable for blunt-end cloning.

The resultant reaction mixture should generally be analyzed bynondenaturing agarose or acrylamide gel electrophoresis to verify thatthe amplification has yielded the predicted product. One would thenprocess the reaction mixture by removing most of the mineral oils,extracting with chloroform to remove the remaining oil, extracting withbuffered phenol and then concentrating by precipitation with 100%ethanol. Next, one should digest about half of the amplified fragmentswith a restriction enzyme that cuts at the flanking sequences used inthe oligonucleotides. The digested fragments are purified on a lowgelling/melting agarose gel.

To subclone the fragments and to check the point mutation, one wouldsubclone the two amplified fragments into an appropriately digestedvector by blunt-end ligation. This would be used to transform E. coli,from which plasmid DNA could subsequently be prepared using a miniprep.The amplified portion of the plasmid DNA would then be analyzed by DNAsequencing to confirm that the correct point mutation was generated.This is important as Taq DNA polymerase can introduce additionalmutations into DNA fragments.

The introduction of a point mutation can also be effected usingsequential PCR™ steps. In this procedure, the two fragments encompassingthe mutation are annealed with each other and extended by mutuallyprimed synthesis. This fragment is then amplified by a second PCR™ step,thereby avoiding the blunt-end ligation required in the above protocol.In this method, the preparation of the template DNA, the generation ofthe oligonucleotide primers and the first PCR™ amplification areperformed as described above. In this process, however, the chosenoligonucleotides should be homologous to the template DNA for a stretchof between about 15 and about 20 bases and must also overlap with eachother by about 10 bases or more.

In the second PCR™ amplification, one would use each amplified fragmentand each flanking sequence primer and carry PCR™ for between about 20and about 25 cycles, using the conditions as described above. One wouldagain subclone the fragments and check that the point mutation wascorrect by using the steps outlined above.

In using either of the foregoing methods, it is generally preferred tointroduce the mutation by amplifying as small a fragment as possible. Ofcourse, parameters such as the melting temperature of theoligonucleotide, as will generally be influenced by the GC content andthe length of the oligo, should also be carefully considered. Theexecution of these methods, and their optimization if necessary, will beknown to those of skill in the art, and are further described in variouspublications, such as Current Protocols in Molecular Biology, 1995,incorporated herein by reference.

When performing site-specific mutagenesis, Table A can be employed as areference.

TABLE A Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

E9. Antibody Fragments

Irrespective of the source of the original anti-aminophospholipidantibody, either the intact antibody, antibody multimers, or any one ofa variety of functional, antigen-binding regions of the antibody may beused in the present invention. Exemplary functional regions includescFv, Fv, Fab′, Fab and F(ab′)₂ fragments of the anti-aminophospholipidantibodies. Techniques for preparing such constructs are well known tothose in the art and are further exemplified herein.

The choice of antibody construct may be influenced by various factors.For example, prolonged half-life can result from the active readsorptionof intact antibodies within the kidney, a property of the Fc piece ofimmunoglobulin. IgG based antibodies, therefore, are expected to exhibitslower blood clearance than their Fab′ counterparts. However, Fab′fragment-based compositions will generally exhibit better tissuepenetrating capability.

Fab fragments can be obtained by proteolysis of the whole immunoglobulinby the non-specific thiol protease, papain. Papain must first beactivated by reducing the sulphydryl group in the active site withcysteine, 2-mercaptoethanol or dithiothreitol. Heavy metals in the stockenzyme should be removed by chelation with EDTA (2 mM) to ensure maximumenzyme activity. Enzyme and substrate are normally mixed together in theratio of 1:100 by weight. After incubation, the reaction can be stoppedby irreversible alkylation of the thiol group with iodoacetamide orsimply by dialysis. The completeness of the digestion should bemonitored by SDS-PAGE and the various fractions separated by proteinA-Sepharose or ion exchange chromatography.

The usual procedure for preparation of F(ab′)₂ fragments from IgG ofrabbit and human origin is limited proteolysis by the enzyme pepsin. Theconditions, 100× antibody excess W/w in acetate buffer at pH 4.5, 37°C., suggest that antibody is cleaved at the C-terminal side of theinter-heavy-chain disulfide bond. Rates of digestion of mouse IgG mayvary with subclass and it may be difficult to obtain high yields ofactive F(ab′)₂ fragments without some undigested or completely degradedIgG. In particular, IgG_(2b) is highly susceptible to completedegradation. The other subclasses require different incubationconditions to produce optimal results, all of which is known in the art.

Digestion of rat IgG by pepsin requires conditions including dialysis in0.1 M acetate buffer, pH 4.5, and then incubation for four hours with 1%w/w pepsin; IgG₁ and IgG_(2a) digestion is improved if first dialyzedagainst 0.1 M formate buffer, pH 2.8, at 4° C., for 16 hours followed byacetate buffer. IgG_(2b) gives more consistent results with incubationin staphylococcal V8 protease (3% w/w) in 0.1 M sodium phosphate buffer,pH 7.8, for four hours at 37° C.

The following patents and patent applications are specificallyincorporated herein by reference for the purposes of even furthersupplementing the present teachings regarding the preparation and use offunctional, antigen-binding regions of antibodies, including scFv, Fv,Fab′, Fab and F(ab′)₂ fragments of the anti-aminophospholipid antibodiesU.S. Pat. Nos. 5,855,866; 5,877,289; 5,965,132; 6,004,555; and6,093,399.

E10. Antibody Conjugates

Anti-aminophospholipid antibodies may be conjugated to anti-cellular orcytotoxic agents, to prepare “immunotoxins”; or operatively associatedwith components that are capable of directly or indirectly stimulatingcoagulation, thus forming a “coaguligand”. In coaguligands, thetargeting agents may be directly linked to a direct or indirectcoagulation factor, or may be linked to a second binding region thatbinds and then releases a direct or indirect coagulation factor. The‘second binding region’ approach generally uses a coagulant-bindingantibody as a second binding region, thus resulting in a bispecificantibody construct. The preparation and use of bispecific antibodies ingeneral is well known in the art, and is further disclosed herein.

In the preparation of immunotoxins, coaguligands and bispecificantibodies, recombinant expression may be employed. The nucleic acidsequences encoding the chosen antibody-based targeting agent areattached, in-frame, to nucleic acid sequences encoding the chosen toxin,coagulant, or second binding region to create an expression unit orvector. Recombinant expression results in translation of the new nucleicacid, to yield the desired protein product. Although antibody-encodingnucleic acids are employed, rather than protein binding ligands, therecombinant approach is essentially the same as those describedhereinabove.

Returning to conjugate technology, the preparation of immunotoxins isgenerally well known in the art. However, certain advantages may beachieved through the application of certain preferred technology, bothin the preparation of the immunotoxins and in their purification forsubsequent clinical administration. For example, while IgG basedimmunotoxins will typically exhibit better binding capability and slowerblood clearance than their Fab′ counterparts, Fab′ fragment-basedimmunotoxins will generally exhibit better tissue penetrating capabilityas compared to IgG based immunotoxins.

Additionally, while numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate the toxinmoiety to the targeting agent, certain linkers will generally bepreferred over other linkers, based on differing pharmacologicalcharacteristics and capabilities. For example, linkers that contain adisulfide bond that is sterically “hindered” are to be preferred, due totheir greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action.

A wide variety of cytotoxic agents are known that may be conjugated toanti-aminophospholipid antibodies, including plant-, fungus- andbacteria-derived toxins, such as ricin A chain or deglycosylated Achain. The cross-linking of a toxin A chain to a targeting agent, incertain cases, requires a cross-linker that presents disulfidefunctions. The reason for this is unclear, but is likely due to a needfor certain toxin moieties to be readily releasable from the targetingagent once the agent has “delivered” the toxin to the targeted cells.

Each type of cross-linker, as well as how the cross-linking isperformed, will tend to vary the pharmacodynamics of the resultantconjugate. Ultimately, in cases where a releasable toxin iscontemplated, one desires to have a conjugate that will remain intactunder conditions found everywhere in the body except the intended siteof action, at which point it is desirable that the conjugate have good“release” characteristics. Therefore, the particular cross-linkingscheme, including in particular the particular cross-linking reagentused and the structures that are cross-linked, will be of somesignificance.

Depending on the specific toxin compound used as part of the fusionprotein, it may be necessary to provide a peptide spacer operativelyattaching the targeting agent and the toxin compound which is capable offolding into a disulfide-bonded loop structure. Proteolytic cleavagewithin the loop would then yield a heterodimeric polypeptide wherein thetargeting agent and the toxin compound are linked by only a singledisulfide bond. An example of such a toxin is a Ricin A-chain toxin.

When certain other toxin compounds are utilized, a non-cleavable peptidespacer may be provided to operatively attach the targeting agent and thetoxin compound of the fusion protein. Toxins which may be used inconjunction with non-cleavable peptide spacers are those which may,themselves, be converted by proteolytic cleavage, into a cytotoxicdisulfide-bonded form. An example of such a toxin compound is aPseudonomas exotoxin compound.

There may be circumstances, such as when the target antigen does notinternalize by a route consistent with efficient intoxication bytargeting agent/toxin compounds, such as immunotoxins, where one willdesire to target chemotherapeutic agents such as anti-tumor drugs, othercytokines, antimetabolites, alkylating agents, hormones, and the like. Avariety of chemotherapeutic and other pharmacological agents have nowbeen successfully conjugated to antibodies and shown to functionpharmacologically. Exemplary antineoplastic agents that have beeninvestigated include doxorubicin, daunomycin, methotrexate, vinblastine,and various others. Moreover, the attachment of other agents such asneocarzinostatin, macromycin, trenimon and α-amanitin has beendescribed.

Where coagulation factors are used in connection with the presentinvention, any covalent linkage to the antibody or targeting agentshould be made at a site distinct from its functional coagulating site.The compositions are thus “linked” in any operative manner that allowseach region to perform its intended function without significantimpairment. Thus, the targeting agents bind to aminophospholipids, andthe coagulation factor promotes blood clotting.

E11. Biochemical Cross-Linkers

In additional to the general information provided above,anti-aminophospholipid antibodies may be conjugated to anti-cellular orcytotoxic agents using certain preferred biochemical cross-linkers.Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different molecules. To link twodifferent proteins in a step-wise manner, hetero-bifunctionalcross-linkers can be used that eliminate unwanted homopolymer formation.Exemplary hetero-bifunctional cross-linkers are referenced in Table B.

TABLE B HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Reactive AdvantagesLength after linker Toward and Applications cross-linking SMPT Primaryamines Greater stability 11.2 A Sulfhydryls SPDP Primary aminesThiolation  6.8 A Sulfhydryls Cleavable cross- linking LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Water-soluble SMCC Primaryamines Stable maleimide 11.6 A Sulfhydryls reactive groupEnzyme-antibody conjugation Hapten-carrier protein conjugationSulfo-SMCC Primary amines Stable maleimide 11.6 A Sulfhydryls reactivegroup Water-soluble Enzyme-antibody conjugation MBS Primary aminesEnzyme-antibody  9.9 A Sulfhydryls conjugation Hapten-carrier proteinconjugation Sulfo-MBS Primary amines Water-soluble  9.9 A SulfhydrylsSIAB Primary amines Enzyme-antibody 10.6 A Sulfhydryls conjugationSulfo-SIAB Primary amines Water-soluble 10.6 A Sulfhydryls SMPB Primaryamines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibodyconjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5 ASulfhydryls Water-soluble EDC/Sulfo-NHS Primary amines Hapten-Carrier  0Carboxyl conjugation groups ABH Carbohydrates Reacts with sugar 11.9 ANonselective groups

Hetero-bifunctional cross-linkers contain two reactive groups: onegenerally reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other generally reacting with a thiol group (e.g.,pyridyl disulfide, maleimides, halogens, etc.). Through the primaryamine reactive group, the cross-linker may react with the lysineresidue(s) of one protein (e.g., the selected antibody or fragment) andthrough the thiol reactive group, the cross-linker, already tied up tothe first protein, reacts with the cysteine residue (free sulfhydrylgroup) of the other protein (e.g., the coagulant).

Compositions therefore generally have, or are derivatized to have, afunctional group available for cross-linking purposes. This requirementis not considered to be limiting in that a wide variety of groups can beused in this manner. For example, primary or secondary amine groups,hydrazide or hydrazine groups, carboxyl alcohol, phosphate, oralkylating groups may be used for binding or cross-linking.

The spacer arm between the two reactive groups of a cross-linkers mayhave various length and chemical compositions. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents). The use of peptide spacers, such asL-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andtoxic or coagulating agents. Linkers that contain a disulfide bond thatis sterically hindered may prove to give greater stability in vivo,preventing release of the agent prior to binding at the site of action.These linkers are thus one preferred group of linking agents.

One of the most preferred cross-linking reagents for use in immunotoxinsis SMPT, which is a bifunctional cross-linker containing a disulfidebond that is “sterically hindered” by an adjacent benzene ring andmethyl groups. It is believed that steric hindrance of the disulfidebond serves a function of protecting the bond from attack by thiolateanions such as glutathione which can be present in tissues and blood,and thereby help in preventing decoupling of the conjugate prior to thedelivery of the attached agent to the tumor site. It is contemplatedthat the SMPT agent may also be used in connection with the bispecificligands of this invention.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers can also beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane. The use of such cross-linkers is wellunderstood in the art.

Once conjugated, the conjugate is separated from unconjugated targetingand therapeutic agents and from other contaminants. A large a number ofpurification techniques are available for use in providing conjugates ofa sufficient degree of purity to render them clinically useful.Purification methods based upon size separation, such as gel filtration,gel permeation or high performance liquid chromatography, will generallybe of most use. Other chromatographic techniques, such as Blue-Sepharoseseparation, may also be used.

E12. Bispecific Antibodies

Bispecific antibodies are particularly useful in the coaguligand aspectsof the present invention. However, bispecific antibodies in general maybe employed, so long as one arm binds to an aminophospholipid and thebispecific antibody is attached to a therapeutic agent, generally at asite distinct from the antigen binding sites. Bispecific antibodies thatbind to both PS and PE may also be used.

In general, the preparation of bispecific antibodies is also well knownin the art. One method involves the separate preparation of antibodieshaving specificity for the targeted antigen, on the one hand, and (asherein) a coagulating agent on the other. Peptic F(ab′γ)₂ fragments areprepared from the two chosen antibodies, followed by reduction of eachto provide separate Fab′γSH fragments. The SH groups on one of the twopartners to be coupled are then alkylated with a cross-linking reagentsuch as o-phenylenedimaleimide to provide free maleimide groups on onepartner. This partner may then be conjugated to the other by means of athioether linkage, to give the desired F(ab′γ)₂ heteroconjugate. Othertechniques are known wherein cross-linking with SPDP or protein A iscarried out, or a trispecific construct is prepared.

Another method for producing bispecific antibodies is by the fusion oftwo hybridomas to form a quadroma. As used herein, the term “quadroma”is used to describe the productive fusion of two B cell hybridomas.Using now standard techniques, two antibody producing hybridomas arefused to give daughter cells, and those cells that have maintained theexpression of both sets of clonotype immunoglobulin genes are thenselected.

A preferred method of generating a quadroma involves the selection of anenzyme deficient mutant of at least one of the parental hybridomas. Thisfirst mutant hybridoma cell line is then fused to cells of a secondhybridoma that had been lethally exposed, e.g., to iodoacetamide,precluding its continued survival. Cell fusion allows for the rescue ofthe first hybridoma by acquiring the gene for its enzyme deficiency fromthe lethally treated hybridoma, and the rescue of the second hybridomathrough fusion to the first hybridoma. Preferred, but not required, isthe fusion of immunoglobulins of the same isotype, but of a differentsubclass. A mixed subclass antibody permits the use if an alternativeassay for the isolation of a preferred quadroma.

In more detail, one method of quadroma development and screeninginvolves obtaining a hybridoma line that secretes the first chosen MAband making this deficient for the essential metabolic enzyme,hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtaindeficient mutants of the hybridoma, cells are grown in the presence ofincreasing concentrations of 8-azaguanine (1×10⁻⁷M to 1×10⁻⁵M). Themutants are subcloned by limiting dilution and tested for theirhypoxanthine/aminopterin/thymidine (HAT) sensitivity. The culture mediummay consist of, for example, DMEM supplemented with 10% FCS, 2 mML-Glutamine and 1 mM penicillin-streptomycin.

A complementary hybridoma cell line that produces the second desired MAbis used to generate the quadromas by standard cell fusion techniques.Briefly, 4.5×10⁷ HAT-sensitive first cells are mixed with 2.8×10⁷HAT-resistant second cells that have been pre-treated with a lethal doseof the irreversible biochemical inhibitor iodoacetamide (5 mM inphosphate buffered saline) for 30 minutes on ice before fusion. Cellfusion is induced using polyethylene glycol (PEG) and the cells areplated out in 96 well microculture plates. Quadromas are selected usingHAT-containing medium. Bispecific antibody-containing cultures areidentified using, for example, a solid phase isotype-specific ELISA andisotype-specific immunofluorescence staining.

In one identification embodiment to identify the bispecific antibody,the wells of microtiter plates (Falcon, Becton Dickinson Labware) arecoated with a reagent that specifically interacts with one of the parenthybridoma antibodies and that lacks cross-reactivity with bothantibodies. The plates are washed, blocked, and the supernatants (SNs)to be tested are added to each well. Plates are incubated at roomtemperature for 2 hours, the supernatants discarded, the plates washed,and diluted alkaline phosphatase-anti-antibody conjugate added for 2hours at room temperature. The plates are washed and a phosphatasesubstrate, e.g., P-Nitrophenyl phosphate (Sigma, St. Louis) is added toeach well. Plates are incubated, 3N NaOH is added to each well to stopthe reaction, and the OD₄₁₀ values determined using an ELISA reader.

In another identification embodiment, microtiter plates pre-treated withpoly-L-lysine are used to bind one of the target cells to each well, thecells are then fixed, e.g. using 1% glutaraldehyde, and the bispecificantibodies are tested for their ability to bind to the intact cell. Inaddition, FACS, immunofluorescence staining, idiotype specificantibodies, antigen binding competition assays, and other methods commonin the art of antibody characterization may be used in conjunction withthe present invention to identify preferred quadromas.

Following the isolation of the quadroma, the bispecific antibodies arepurified away from other cell products. This may be accomplished by avariety of protein isolation procedures, known to those skilled in theart of immunoglobulin purification. Means for preparing andcharacterizing antibodies are well known in the art (See, e.g.,Antibodies: A Laboratory Manual, 1988).

For example, supernatants from selected quadromas are passed overprotein A or protein G sepharose columns to bind IgG (depending on theisotype). The bound antibodies are then eluted with, e.g. a pH 5.0citrate buffer. The elute fractions containing the BsAbs, are dialyzedagainst an isotonic buffer. Alternatively, the eluate is also passedover an anti-immunoglobulin-sepharose column. The BsAb is then elutedwith 3.5 M magnesium chloride. BsAbs purified in this way are thentested for binding activity by, e.g., an isotype-specific ELISA andimmunofluorescence staining assay of the target cells, as describedabove.

Purified BsAbs and parental antibodies may also be characterized andisolated by SDS-PAGE electrophoresis, followed by staining with silveror Coomassie. This is possible when one of the parental antibodies has ahigher molecular weight than the other, wherein the band of the BsAbsmigrates midway between that of the two parental antibodies. Reductionof the samples verifies the presence of heavy chains with two differentapparent molecular weights.

F. Pharmaceutical Compositions

The most basic pharmaceutical compositions of the present invention willgenerally comprise an effective amount of at least a first therapeuticagent-targeting agent construct, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Combinedtherapeutics are also contemplated, and the same type of underlyingpharmaceutical compositions may be employed for both single and combinedmedicaments.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. For humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards. Supplementary active ingredients can also beincorporated into the compositions.

F1. Parenteral Formulations

The therapeutic agent-targeting agent constructs of the presentinvention will most often be formulated for parenteral administration,e.g., formulated for injection via the intravenous, intramuscular,sub-cutaneous, transdermal, or other such routes, including peristalticadministration and direct instillation into a tumor or disease site(intracavity administration). The preparation of an aqueous compositionthat contains a therapeutic agent-targeting agent construct as an activeingredient will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and fluid to theextent that syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

The therapeutic agent-targeting agent compositions can be formulatedinto a sterile aqueous composition in a neutral or salt form. Solutionsof the therapeutic agent-targeting agents as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein), and those that areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, trifluoroacetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

Suitable carriers include solvents and dispersion media containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants.

Under ordinary conditions of storage and use, all such preparationsshould contain a preservative to prevent the growth of microorganisms.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Prior to or upon formulation, the therapeutic agent-targeting agentconstructs should be extensively dialyzed to remove undesired smallmolecular weight molecules, and/or lyophilized for more readyformulation into a desired vehicle, where appropriate. Sterileinjectable solutions are prepared by incorporating the activetherapeutic agent-targeting agents in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as desired, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle that contains the basic dispersionmedium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques that yield a powder of the active therapeuticagent-targeting agent ingredient, plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the therapeutic agent-targetingagent construct admixed with an acceptable pharmaceutical diluent orexcipient, such as a sterile aqueous solution, to give a range of finalconcentrations, depending on the intended use. The techniques ofpreparation are generally well known in the art as exemplified byRemington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company,1980, incorporated herein by reference. It should be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

Upon formulation, therapeutic agent-targeting agent solutions will beadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically effective. Formulations are easilyadministered in a variety of dosage forms, such as the type ofinjectable solutions described above, but other pharmaceuticallyacceptable forms are also contemplated, e.g., tablets, pills, capsulesor other solids for oral administration, suppositories, pessaries, nasalsolutions or sprays, aerosols, inhalants, liposomal forms and the like.Pharmaceutical “slow release” capsules or compositions may also be used.Slow release formulations are generally designed to give a constant druglevel over an extended period and may be used to deliver therapeuticagent-targeting agent constructs in accordance with the presentinvention.

F2. Liposomes and Nanocapsules

In certain embodiments, liposomes and/or nanoparticles may also beemployed with the therapeutic agent-targeting agent constructs. Theformation and use of liposomes is generally known to those of skill inthe art, as summarized below.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on pH, ionic strength and thepresence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

G. Therapeutic Kits

This invention also provides therapeutic kits comprising therapeuticagent-targeting agent constructs for use in the present treatmentmethods. Such kits will generally contain, in suitable container means,a pharmaceutically acceptable formulation of at least one therapeuticagent-targeting agent construct. The kits may also contain otherpharmaceutically acceptable formulations, either for diagnosis/imagingor combined therapy. For example, such kits may contain any one or moreof a range of chemotherapeutic or radiotherapeutic drugs;anti-angiogenic agents; anti-tumor cell antibodies; and/or anti-tumorvasculature or anti-tumor stroma immunotoxins or coaguligands.

The kits may have a single container (container means) that contains thetherapeutic agent-targeting agent construct, with or without anyadditional components, or they may have distinct containers for eachdesired agent. Where combined therapeutics are provided, a singlesolution may be pre-mixed, either in a molar equivalent combination, orwith one component in excess of the other. Alternatively, each of thetherapeutic agent-targeting agent construct and other anti-cancer agentcomponents of the kit may be maintained separately within distinctcontainers prior to administration to a patient.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is preferably an aqueous solution, with asterile aqueous solution being particularly preferred. However, thecomponents of the kit may be provided as dried powder(s). When reagentsor components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container.

The containers of the kit will generally include at least one vial, testtube, flask, bottle, syringe or other container means, into which thetherapeutic agent-targeting agent construct, and any other desiredagent, may be placed and, preferably, suitably aliquoted. Where separatecomponents are included, the kit will also generally contain a secondvial or other container into which these are placed, enabling theadministration of separated designed doses. The kits may also comprise asecond/third container means for containing a sterile, pharmaceuticallyacceptable buffer or other diluent.

The kits may also contain a means by which to administer the therapeuticagent-targeting agent construct to an animal or patient, e.g., one ormore needles or syringes, or even an eye dropper, pipette, or other suchlike apparatus, from which the formulation may be injected into theanimal or applied to a diseased area of the body. The kits of thepresent invention will also typically include a means for containing thevials, or such like, and other component, in close confinement forcommercial sale, such as, e.g., injection or blow-molded plasticcontainers into which the desired vials and other apparatus are placedand retained.

H. Tumor Treatment

The most important use of the present invention is in the treatment ofvascularized, malignant tumors; with the treatment of benign tumors,such as BPH, also being contemplated. The invention may also be used inthe therapy of other diseases and disorders having, as a component ofthe disease, prothrombotic blood vessels. Such vasculature-associateddiseases include diabetic retinopathy, macular degeneration, vascularrestenosis, including restenosis following angioplasty, arteriovenousmalformations (AVM), meningioma, hemangioma, neovascular glaucoma andpsoriasis; and also angiofibroma, arthritis, rheumatoid arthritis,atherosclerotic plaques, corneal graft neovascularization, hemophilicjoints, hypertrophic scars, osler-weber syndrome, pyogenic granulomaretrolental fibroplasia, scleroderma, trachoma, vascular adhesions,synovitis, dermatitis, various other inflammatory diseases anddisorders, and even endometriosis.

The therapeutic agent-targeting agent construct treatment of theinvention is most preferably exploited for the treatment of solidtumors. Such uses may employ therapeutic agent-targeting agentconstructs alone or in combination with chemotherapeutic,radiotherapeutic, apoptopic, anti-angiogenic agents and/or immunotoxinsor coaguligands. The therapeutic agent-targeting agent construct methodsprovided by this invention are broadly applicable to the treatment ofany malignant tumor having a vascular component. Typical vascularizedtumors are the solid tumors, particularly carcinomas, which require avascular component for the provision of oxygen and nutrients. Exemplarysolid tumors that may be treated using the invention include, but arenot limited to, carcinomas of the lung, breast, ovary, stomach,pancreas, larynx, esophagus, testes, liver, parotid, biliary tract,colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate,thyroid, squamous cell carcinomas, adenocarcinomas, small cellcarcinomas, melanomas, gliomas, neuroblastomas, and the like.

The present invention is contemplated for use in the treatment of anypatient that presents with a solid tumor. However, in that thisinvention is particularly successful in the treatment of solid tumors ofmoderate or large sizes, patients in these categories are likely toreceive more significant benefits from treatment in accordance with themethods and compositions provided herein.

Therefore, in general, the invention can be used to treat tumors ofabout 0.3–0.5 cm and upwards, although it is a better use of theinvention to treat tumors of greater than 0.5 cm in size. From thestudies already conducted in acceptable animal models, it is believedthat patients presenting with tumors of between about 1.0 and about 2.0cm in size will be in the preferred treatment group of patients fortherapeutic agent-targeting agent therapy, although tumors up to andincluding the largest tumors found in humans may also be treated.

Although the present invention is not generally intended as apreventative or prophylactic treatment, use of the invention iscertainly not confined to the treatment of patients having tumors ofonly moderate or large sizes. There are many reasons underlying thisaspect of the breadth of the invention. For example, a patientpresenting with a primary tumor of moderate size or above may also havevarious other metastatic tumors that are considered to be small-sized oreven in the earlier stages of metastatic tumor seeding. Given that thetherapeutic agent-targeting agent constructs, or combinations, of theinvention are generally administered into the systemic circulation of apatient, they will naturally have effects on the secondary, smaller andmetastatic tumors, although this may not be the primary intent of thetreatment. Furthermore, even in situations where the tumor mass as awhole is a single small tumor, certain beneficial anti-tumor effectswill result from the use of the present therapeutic agent-targetingagent treatment.

The guidance provided herein regarding the most suitable patients foruse in connection with the present invention is intended as teachingthat certain patient's profiles may assist with the selection ofpatients for treatment by the present invention. The pre-selection ofcertain patients, or categories of patients, does not in any way negatethe basic usefulness of the present invention in connection with thetreatment of all patients having a vascularized tumor. A furtherconsideration is the fact that the assault on the tumor provided by thetherapeutic agent-targeting agent construct of the invention maypredispose the tumor to further therapeutic treatment, such that thesubsequent treatment results in an overall synergistic effect or evenleads to total remission or cure.

It is not believed that any particular type of tumor should be excludedfrom treatment using the present invention. However, the type of tumorcells may be relevant to the use of the invention in combination withsecondary therapeutic agents, particularly chemotherapeutics andanti-tumor cell immunotoxins. As the effect of the present therapy is todestroy the tumor vasculature, and as the vasculature is substantiallyor entirely the same in all solid tumors, it will be understood that thepresent therapeutic agent-targeting agent methodology is widely orentirely applicable to the treatment of all solid tumors, irrespectiveof the particular phenotype or genotype of the tumor cells themselves.

Therapeutically effective doses of therapeutic agent-targeting agentconstructs are readily determinable using data from an animal model, asshown in the studies detailed herein. Experimental animals bearing solidtumors are frequently used to optimize appropriate therapeutic dosesprior to translating to a clinical environment. Such models are known tobe very reliable in predicting effective anti-cancer strategies. Forexample, mice bearing solid tumors, such as used in the Examples, arewidely used in pre-clinical testing. The inventors have used suchart-accepted mouse models to determine working ranges of therapeuticagent-targeting agent constructs that give beneficial anti-tumor effectswith minimal toxicity.

As is known in the art, there are realistic objectives that may be usedas a guideline in connection with pre-clinical testing before proceedingto clinical treatment. However, due to the safety already demonstratedin accepted models, pre-clinical testing of the present invention willbe more a matter of optimization, rather than to confirm effectiveness.Thus, pre-clinical testing may be employed to select the mostadvantageous therapeutic agent-targeting agent constructs, doses orcombinations.

Any therapeutic agent-targeting agent dose, or combined medicament, thatresults in any consistent detectable tumor vasculature destruction,thrombosis and anti-tumor effects will still define a useful invention.Destructive, thrombotic and necrotic effects should be observed inbetween about 10% and about 40–50% of the tumor blood vessels and tumortissues, upwards to between about 50% and about 99% of such effectsbeing observed. The present invention may also be effective againstvessels downstream of the tumor, i.e., target at least a sub-set of thedraining vessels, particularly as cytokines released from the tumor willbe acting on these vessels, changing their antigenic profile.

It will also be understood that even in such circumstances where theanti-tumor effects of the therapeutic agent-targeting agent dose, orcombined therapy, are towards the low end of this range, it may be thatthis therapy is still equally or even more effective than all otherknown therapies in the context of the particular tumor targets. It isunfortunately evident to a clinician that certain tumors cannot beeffectively treated in the intermediate or long term, but that does notnegate the usefulness of the present therapy, particularly where it isat least about as effective as the other strategies generally proposed.

In designing appropriate doses of therapeutic agent-targeting agentconstructs, or combined therapeutics, for the treatment of vascularizedtumors, one may readily extrapolate from the animal studies describedherein in order to arrive at appropriate doses for clinicaladministration. To achieve this conversion, one would account for themass of the agents administered per unit mass of the experimental animaland, preferably, account for the differences in the body surface areabetween the experimental animal and the human patient. All suchcalculations are well known and routine to those of ordinary skill inthe art.

For example, in taking the successful doses of annexin-TF constructs inthe mouse studies, and applying standard calculations based upon massand surface area, effective doses for use in human patients would bebetween about 1 mg and about 500 mgs antibody per patient, andpreferably, between about 10 mgs and about 100 mgs antibody per patient.

Accordingly, using this information, the inventors contemplate thatuseful low doses of therapeutic agent-targeting agent constructs forhuman administration will be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or about 30 mgs or so per patient; and useful high doses oftherapeutic agent-targeting agent constructs for human administrationwill be about 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or about500 mgs or so per patient. Useful intermediate doses of therapeuticagent-targeting agent constructs for human administration arecontemplated to be about 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175,200 or about 225 mgs or so per patient.

Any particular range using any of the foregoing recited exemplary dosesor any value intermediate between the particular stated ranges iscontemplated. It will also be understood that therapeuticagent-targeting agent constructs with coagulants can generally be usedat higher doses than those with toxins.

In general, dosage ranges of between about 5–100 mgs, about 10–80 mgs,about 20–70 mgs, about 25–60 mgs, or about 30–50 mgs or so oftherapeutic agent-targeting agent construct per patient will bepreferred. Notwithstanding these stated ranges, it will be understoodthat, given the parameters and detailed guidance presented herein,further variations in the active or optimal ranges will be encompassedwithin the present invention. Although doses in and around about 5 or 10to about 70, 80, 90 or 100 mgs per patient are currently preferred, itwill be understood that lower doses may be more appropriate incombination with other agents, and that high doses can still betolerated, particularly given the enhanced safety of the coagulantconstructs. The use of human or humanized antibodies or binding proteinsrenders the present invention even safer for clinical use, furtherreducing the chances of significant toxicity or side effects in healthytissues.

The intention of the therapeutic regimens of the present invention isgenerally to produce significant anti-tumor effects whilst still keepingthe dose below the levels associated with unacceptable toxicity. Inaddition to varying the dose itself, the administration regimen can alsobe adapted to optimize the treatment strategy. A currently preferredtreatment strategy is to administer between about 1–500 mgs, andpreferably, between about 10–100 mgs of the therapeutic agent-targetingagent construct, or therapeutic cocktail containing such, about 3 timeswithin about a 7 day period. For example, doses would be given on aboutday 1, day 3 or 4 and day 6 or 7.

In administering the particular doses themselves, one would preferablyprovide a pharmaceutically acceptable composition (according to FDAstandards of sterility, pyrogenicity, purity and general safety) to thepatient systemically. Intravenous injection is generally preferred, andthe most preferred method is to employ a continuous infusion over a timeperiod of about 1 or 2 hours or so. Although it is not required todetermine such parameters prior to treatment using the presentinvention, it should be noted that the studies detailed herein result inat least some thrombosis being observed specifically in the bloodvessels of a solid tumor within about 12–24 hours of injection, and thatwidespread tumor necrosis is also observed in this period.

Naturally, before wide-spread use, clinical trials will be conducted.The various elements of conducting a clinical trial, including patienttreatment and monitoring, will be known to those of skill in the art inlight of the present disclosure. The following information is beingpresented as a general guideline for use in establishing such trials.

Patients chosen for the first therapeutic agent-targeting agentconstruct treatment studies will have failed to respond to at least onecourse of conventional therapy, and will have objectively measurabledisease as determined by physical examination, laboratory techniques,and/or radiographic procedures. Any chemotherapy should be stopped atleast 2 weeks before entry into the study. Where murine monoclonalantibodies or antibody portions are employed, the patients should haveno history of allergy to mouse immunoglobulin.

Certain advantages will be found in the use of an indwelling centralvenous catheter with a triple lumen port. The therapeuticagent-targeting agent constructs should be filtered, for example, usinga 0.22μ filter, and diluted appropriately, such as with saline, to afinal volume of 100 ml. Before use, the test sample should also befiltered in a similar manner, and its concentration assessed before andafter filtration by determining the A₂₈₀. The expected recovery shouldbe within the range of 87% to 99%, and adjustments for protein loss canthen be accounted for.

The therapeutic agent-targeting agent constructs may be administeredover a period of approximately 4–24 hours, with each patient receiving2–4 infusions at 2–7 day intervals. Administration can also be performedby a steady rate of infusion over a 7 day period. The infusion given atany dose level should be dependent upon any toxicity observed. Hence, ifGrade II toxicity was reached after any single infusion, or at aparticular period of time for a steady rate infusion, further dosesshould be withheld or the steady rate infusion stopped unless toxicityimproved. Increasing doses of therapeutic agent-targeting agentconstructs should be administered to groups of patients untilapproximately 60% of patients showed unacceptable Grade III or IVtoxicity in any category. Doses that are ⅔ of this value are defined asthe safe dose.

Physical examination, tumor measurements, and laboratory tests should,of course, be performed before treatment and at intervals up to 1 monthlater. Laboratory tests should include complete blood counts, serumcreatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT,bilirubin, albumin, and total serum protein. Serum samples taken up to60 days after treatment should be evaluated by radioimmunoassay for thepresence of the administered therapeutic agent-targeting agentconstructs, and antibodies against any portions thereof. Immunologicalanalyses of sera, using any standard assay such as, for example, anELISA or RIA, will allow the pharmacokinetics and clearance of theanti-aminophospholipid therapeutic agent to be evaluated.

To evaluate the anti-tumor responses, the patients should be examined at48 hours to 1 week and again at 30 days after the last infusion. Whenpalpable disease was present, two perpendicular diameters of all massesshould be measured daily during treatment, within 1 week aftercompletion of therapy, and at 30 days. To measure nonpalpable disease,serial CT scans could be performed at 1-cm intervals throughout thechest, abdomen, and pelvis at 48 hours to 1 week and again at 30 days.Tissue samples should also be evaluated histologically, and/or by flowcytometry, using biopsies from the disease sites or even blood or fluidsamples if appropriate.

Clinical responses may be defined by acceptable measure. For example, acomplete response may be defined by the disappearance of all measurabletumor 1 month after treatment. Whereas a partial response may be definedby a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules 1 month aftertreatment, with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater 1 month aftertreatment, with progression in one or more sites.

In light of results from clinical trials, such as those described above,an even more precise treatment regimen may be formulated. Even so, somevariation in dosage may later be necessary depending on the condition ofthe subject being treated. The physician responsible for administrationwill, in light of the present disclosure, be able to determine theappropriate dose for the individual subject. Such optimization andadjustment is routinely carried out in the art and by no means reflectsan undue amount of experimentation.

I. Tumor Imaging

The present invention further provides combined tumor treatment andimaging methods, based upon anti-aminophospholipid binding ligands.Anti-aminophospholipid binding proteins or antibodies that are linked toone or more detectable agents are envisioned for use in pre-imaging thetumor, forming a reliable image prior to the treatment, which itselftargets the aminophospholipid markers.

The anti-aminophospholipid imaging ligands or antibodies, or conjugatesthereof, will generally comprise an anti-aminophospholipid antibody orbinding ligand operatively attached, or conjugated to, a detectablelabel. “Detectable labels” are compounds or elements that can bedetected due to their specific functional properties, or chemicalcharacteristics, the use of which allows the component to which they areattached to be detected, and further quantified if desired. Preferably,the detectable labels are those detectable in vivo using non-invasivemethods.

Antibody and binding protein conjugates for use as diagnostic agentsgenerally fall into two classes, those for use in in vitro diagnostics,such as in a variety of immunoassays, and those for use in vivodiagnostic protocols. It is the in vivo imaging methods that areparticularly intended for use with this invention.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies and binding ligands (see, e.g., U.S. Pat.Nos. 5,021,236 and 4,472,509, both incorporated herein by reference).Certain attachment methods involve the use of a metal chelate complexemploying, for example, an organic chelating agent such a DTPA attachedto the antibody (U.S. Pat. No. 4,472,509). Monoclonal antibodies mayalso be reacted with an enzyme in the presence of a coupling agent suchas glutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate.

An example of detectable labels are the paramagnetic ions. In this case,suitable ions include chromium (III), manganese (II), iron (III), iron(II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium(III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred.

Ions useful in other contexts, such as X-ray imaging, include but arenot limited to lanthanum (III), gold (III), lead (II), and especiallybismuth (III). Fluorescent labels include rhodamine, fluorescein andrenographin. Rhodamine and fluorescein are often linked via anisothiocyanate intermediate.

In the case of radioactive isotopes for diagnostic applications,suitable examples include ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt,⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵,iodine ³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium ¹⁸⁸,⁷⁵selenium, ³⁵sulphur, technetium^(99m) and yttrium⁹⁰. ¹²⁵I is oftenbeing preferred for use in certain embodiments, and technicium^(99m) andindium¹¹¹ are also often preferred due to their low energy andsuitability for long range detection.

Radioactively labeled anti-aminophospholipid antibodies and bindingligands for use in the present invention may be produced according towell-known methods in the art. For instance, intermediary functionalgroups that are often used to bind radioisotopic metallic ions toantibodies are diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetracetic acid (EDTA).

Monoclonal antibodies can also be iodinated by contact with sodium orpotassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.Anti-aminophospholipid antibodies according to the invention may belabeled with technetium-99m by ligand exchange process, for example, byreducing pertechnate with stannous solution, chelating the reducedtechnetium onto a Sephadex column and applying the antibody to thiscolumn; or by direct labeling techniques, e.g., by incubatingpertechnate, a reducing agent such as SNCl₂, a buffer solution such assodium-potassium phthalate solution, and the antibody.

Any of the foregoing type of detectably labeled anti-aminophospholipidantibodies and aminophospholipid binding ligands may be used in theimaging aspects of the present invention. Although not previouslyproposed for use in combined tumor imaging and treatment, thedetectably-labeled annexins of U.S. Pat. No. 5,627,036; WO 95/19791; WO95/27903; WO 95/34315; WO 96/17618; and WO 98/04294; each incorporatedherein by reference; may also be employed.

WO 95/27903 (incorporated herein by reference) provides annexins for usein detecting apoptotic cells. Any of the annexin-detectable agentmarkers of WO 95/27903 may be used herein, although it will be knownthat certain of these are more suitable for in vitro uses. WO 95/27903is also specifically incorporated herein by reference for purposes ofproviding detectable kits that may be adapted for combined use with thetherapeutics of the present invention.

Each of WO 95/19791; WO 95/34315; WO 96/17618; and WO 98/04294; are alsoincorporated herein by reference for purposes of further describingradiolabelled annexin conjugates for diagnostic imaging. The intent ofeach of the foregoing documents is to provide radiolabelled annexins foruse in imaging vascular thromboses, particularly in or near the heart,such as in deep vein thrombosis, pulmonary embolism, myocardialinfarction, atrial fibrillation, problems with prosthetic cardiovascularmaterials, stroke, and the like. These radiolabelled annexins were alsoproposed for use in imaging activated platelets, e.g., in conditionssuch as abscesses, restenosis, inflammation of joints, clots in cerebralarteries, etc.

U.S. Pat. No. 5,627,036 (incorporated herein by reference) alsogenerally concerns ‘annexine’ (annexin) binding ligands for use inanalyzing platelet phosphatidylserine. It is explained in U.S. Pat. No.5,627,036 that hemostatic disorders, such as arterial, coronary andvenous thrombosis, are usually idiopathic, which makes prediction andprevention difficult. To recognize such hemostatic disorders earlier,the detection of activated platelets is proposed. The detectably labeledannexins compositions are thus disclosed in order to detect activatedplatelets in hemostatic disorders (U.S. Pat. No. 5,627,036).

Although proposing a wide range of diagnostic uses, none of WO 95/19791;WO 95/34315; WO 96/17618; or WO 98/04294 make reference to imaging thevasculature of solid tumors. Neither does U.S. Pat. No. 5,627,036 makeany such suggestions. Nonetheless, the disclosed detectable andradiolabelled annexin compositions per se may now be used to advantagein this regard, in light of the surprising discoveries disclosed herein.

In particular, U.S. Pat. No. 5,627,036 (incorporated herein byreference) discloses annexins detectably labeled with fluoresceinisothiocyanate; radioisotopes of halogens, technetium, lead, mercury,thallium or indium; and paramagnetic contrast agents.

WO 95/19791 (incorporated herein by reference) provides conjugates ofannexin bonded to an N₂S₂ chelate that can be radiolabelled bycomplexing a radionuclide to the chelate. WO 95/34315 (incorporatedherein by reference) provides annexin conjugates comprising one or moregalactose residues with the N₂S₂ chelate. The galactose moiety is saidto facilitate the rapid elimination of the radiolabelled conjugate fromthe circulation, reducing radiation damage to non-target tissues andbackground ‘noise.’

WO 96/17618 (incorporated herein by reference) in turn provides annexinconjugates suitable for radiolabeling with diagnostic imaging agentsthat comprise an annexin with a cluster of galactose residues and anN₂S₂ chelate. These are reported to have a shorter circulating half-lifeand a higher binding affinity for target sites than the foregoingradiolabeled annexin-galactose conjugates.

Still further radiolabeled annexin conjugates are provided by WO98/04294 (incorporated herein by reference). These conjugates comprisean annexin that is modified to provide an accessible sulphydryl groupconjugated to a hexose moiety that is recognized by a mammalian liverreceptor. Annexin multimer conjugates and chelating compounds conjugatedvia esterase-sensitive bonds are also provided.

Each of WO 95/19791; WO 95/34315; WO 96/17618; and WO 98/04294; are alsospecifically incorporated herein by reference for purposes of providingannexin conjugate components for radiolabelling that are amenable topackaging in “cold kits”, i.e., wherein the components are provided inseparate vials. U.S. Pat. No. 5,627,036 similarly provides kitscomprising a carrier being compartmentalized to receive detectablylabeled annexins that may be adapted for use herewith.

Although suitable for use in in vitro diagnostics, the presentaminophospholipid detection methods are more intended for forming animage of the tumor vasculature of a patient prior to treatment withtherapeutic agent-targeting agent constructs. The in vivo diagnostic orimaging methods generally comprise administering to a patient adiagnostically effective amount of an anti-aminophospholipid antibody orbinding ligand that is conjugated to a marker that is detectable bynon-invasive methods. The antibody- or binding ligand-marker conjugateis allowed sufficient time to localize and bind to the aminophospholipidexpressed on the luminal surface of the tumor vasculature. The patientis then exposed to a detection device to identify the detectable marker,thus forming an image of the tumor vasculature.

The nuclear magnetic spin-resonance isotopes, such as gadolinium, aredetected using a nuclear magnetic imaging device; and radioactivesubstances, such as technicium^(99m) or indium¹¹¹, are detected using agamma scintillation camera or detector. U.S. Pat. No. 5,627,036 is alsospecifically incorporated herein by reference for purposes of providingeven further guidance regarding the safe and effective introduction ofsuch detectably labeled constructs into the blood of an individual, andmeans for determining the distribution of the detectably labeled annexinextracorporally, e.g., using a gamma scintillation camera or by magneticresonance measurement.

Dosages for imaging embodiments are generally less than for therapy, butare also dependent upon the age and weight of a patient. A one time doseof between about 0.1, 0.5 or about 1 mg and about 9 or 10 mgs, and morepreferably, of between about 1 mg and about 5–10 mgs ofanti-aminophospholipid antibody- or aminophospholipid bindingligand-conjugate per patient is contemplated to be useful. U.S. Pat. No.5,627,036; and WO 95/19791, each incorporated herein by reference, arealso instructive regarding doses of detectably-labeled annexins.

J. Combination Therapies

The therapeutic agent-targeting agent treatment methods of the presentinvention may be combined with any other methods generally employed inthe treatment of the particular tumor, disease or disorder that thepatient exhibits. So long as a particular therapeutic approach is notknown to be detrimental to the patient's condition in itself, and doesnot significantly counteract the therapeutic agent-targeting agenttreatment, its combination with the present invention is contemplated.

In connection solid tumor treatment, the present invention may be usedin combination with classical approaches, such as surgery, radiotherapy,chemotherapy, and the like. The invention therefore provides combinedtherapies in which therapeutic agent-targeting agent constructs are usedsimultaneously with, before, or after surgery or radiation treatment; orare administered to patients with, before, or after conventionalchemotherapeutic, radiotherapeutic or anti-angiogenic agents, ortargeted immunotoxins or coaguligands.

Combination therapy for other vascular diseases is also contemplated. Aparticular example of such is benign prostatic hyperplasia (BPH), whichmay be treated with therapeutic agent-targeting agent constructs incombination other treatments currently practiced in the art. Forexample, targeting of immunotoxins to markers localized within BPH, suchas PSA.

When one or more agents are used in combination with the therapeuticagent-targeting agent therapy, there is no requirement for the combinedresults to be additive of the effects observed when each treatment isconducted separately. Although at least additive effects are generallydesirable, any increased anti-tumor effect above one of the singletherapies would be of benefit. Also, there is no particular requirementfor the combined treatment to exhibit synergistic effects, although thisis certainly possible and advantageous.

To practice combined anti-tumor therapy, one would simply administer toan animal a therapeutic agent-targeting agent construct in combinationwith another anti-cancer agent in a manner effective to result in theircombined anti-tumor actions within the animal. The agents wouldtherefore be provided in amounts effective and for periods of timeeffective to result in their combined presence within the tumorvasculature and their combined actions in the tumor environment. Toachieve this goal, the therapeutic agent-targeting agent constructs andanti-cancer agents may be administered to the animal simultaneously,either in a single composition, or as two distinct compositions usingdifferent administration routes.

Alternatively, the therapeutic agent-targeting agent treatment mayprecede, or follow, the anti-cancer agent treatment by, e.g., intervalsranging from minutes to weeks. In certain embodiments where theanti-cancer agent and therapeutic agent-targeting agent construct areapplied separately to the animal, one would ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the anti-cancer agent and therapeutic agent-targeting agentcomposition would still be able to exert an advantageously combinedeffect on the tumor. In such instances, it is contemplated that onewould contact the tumor with both agents within about 5 minutes to aboutone week of each other and, more preferably, within about 12–72 hours ofeach other, with a delay time of only about 12–48 hours being mostpreferred.

Exemplary anti-cancer agents that would be given prior to thetherapeutic agent-targeting agent construct are agents that induce theexpression of aminophospholipids within the tumor vasculature. Forexample, agents that stimulate localized calcium production and/or thatinduce apoptosis will generally result in increased PS expression, whichcan then be targeted using a subsequent anti-PS therapeuticagent-targeting agent construct. Therapeutic agent-targeting agentconstructs would be first administered in other situations to causetumor destruction, followed by, e.g., anti-angiogenic therapies ortherapies directed to targeting necrotic tumor cells.

The general use of combinations of substances in cancer treatment iswell know. For example, U.S. Pat. No. 5,710,134 (incorporated herein byreference) discloses components that induce necrosis in tumors incombination with non-toxic substances or “prodrugs”. The enzymes setfree by necrotic processes cleave the non-toxic “prodrug” into the toxic“drug”, which leads to tumor cell death. Also, U.S. Pat. No. 5,747,469(incorporated herein by reference) discloses the combined use of viralvectors encoding p53 and DNA damaging agents. Any such similarapproaches can be used with the present invention.

In some situations, it may even be desirable to extend the time periodfor treatment significantly, where several days (2, 3, 4, 5, 6 or 7),several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Thiswould be advantageous in circumstances where one treatment was intendedto substantially destroy the tumor, such as the therapeuticagent-targeting agent treatment, and another treatment was intended toprevent micrometastasis or tumor re-growth, such as the administrationof an anti-angiogenic agent. The EN 7/44 antibody of Hagemeier et al.(1986) is not believed to be an effective anti-angiogenic agent, lackingbinding to a surface accessible antigen, amongst other deficiencies.

It also is envisioned that more than one administration of either thetherapeutic agent-targeting agent construct or the anti-cancer agentwill be utilized. The therapeutic agent-targeting agent constructs andanti-cancer agents may be administered interchangeably, on alternatedays or weeks; or a sequence of therapeutic agent-targeting agenttreatment may be given, followed by a sequence of anti-cancer agenttherapy. In any event, to achieve tumor regression using a combinedtherapy, all that is required is to deliver both agents in a combinedamount effective to exert an anti-tumor effect, irrespective of thetimes for administration.

In terms of surgery, any surgical intervention may be practiced incombination with the present invention. In connection with radiotherapy,any mechanism for inducing DNA damage locally within tumor cells iscontemplated, such as γ-irradiation, X-rays, UV-irradiation, microwavesand even electronic emissions and the like. The directed delivery ofradioisotopes to tumor cells is also contemplated, and this may be usedin connection with a targeting antibody or other targeting means.

Cytokine therapy also has proven to be an effective partner for combinedtherapeutic regimens. Various cytokines may be employed in such combinedapproaches. Examples of cytokines include IL-1α IL-1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-β, GM-CSF,M-CSF, G-CSF, TNFα, TNFβ, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM,TMF, PDGF, IFN-α, IFN-β, IFN-γ. Cytokines are administered according tostandard regimens, consistent with clinical indications such as thecondition of the patient and relative toxicity of the cytokine.Uteroglobins may also be used to prevent or inhibit metastases (U.S.Pat. No. 5,696,092; incorporated herein by reference).

J1. Chemotherapeutics

In certain embodiments, the therapeutic agent-targeting agent constructsof the present invention may be administered in combination with achemotherapeutic agent. Chemotherapeutic drugs can kill proliferatingtumor cells, enhancing the necrotic areas created by the overalltreatment. The drugs can thus enhance the thrombotic action of thetherapeutic agent-targeting agent constructs.

By inducing the formation of thrombi in tumor vessels, the therapeuticagent-targeting agent constructs can enhance the action of thechemotherapeutics by retaining or trapping the drugs within the tumor.The chemotherapeutics are thus retained within the tumor, while the restof the drug is cleared from the body. Tumor cells are thus exposed to ahigher concentration of drug for a longer period of time. Thisentrapment of drug within the tumor makes it possible to reduce the doseof drug, making the treatment safer as well as more effective.

Irrespective of the underlying mechanism(s), a variety ofchemotherapeutic agents may be used in the combined treatment methodsdisclosed herein. Chemotherapeutic agents contemplated as exemplaryinclude, e.g., tamoxifen, taxol, vincristine, vinblastine, etoposide(VP-16), adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D,mitomycin C, cisplatin (CDDP), combretastatin(s) and derivatives andprodrugs thereof.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. By way of example only, agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Further useful agents include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25–75 mg/m²at 21 day intervals for adriamycin, to 35–50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used.

Exemplary chemotherapeutic agents that are useful in connection withcombined therapy are listed in Table C. Each of the agents listedtherein are exemplary and by no means limiting. The skilled artisan isdirected to “Remington's Pharmaceutical Sciences” 15th Edition, chapter33, in particular pages 624–652. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The physician responsible for administration will be able todetermine the appropriate dose for the individual subject.

TABLE C CHEMOTHERAPEUTIC AGENTS USEFUL IN NEOPLASTIC DISEASENONPROPRIETARY NAMES CLASS TYPE OF AGENT (OTHER NAMES) DISEASEAlkylating Agents Nitrogen Mustards Mechlorethamine (HN₂) Hodgkin'sdisease, non-Hodgkin's lymphomas Cyclophosphamide Acute and chroniclymphocytic Ifosfamide leukemias, Hodgkin's disease, non- Hodgkin'slymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung, Wilms'tumor, cervix, testis, soft-tissue sarcomas Melphalan (L-sarcolysin)Multiple myeloma, breast, ovary Chlorambucil Chronic lymphocyticleukemia, primary macroglobulinemia, Hodgkin's disease, non-Hodgkin'slymphomas Ethylenimenes and Hexamethylmelamine Ovary MethylmelaminesThiotepa Bladder, breast, ovary Alkyl Sulfonates Busulfan Chronicgranulocytic leukemia Nitrosoureas Carmustine (BCNU) Hodgkin's disease,non-Hodgkin's lymphomas, primary brain tumors, multiple myeloma,malignant melanoma Lomustine (CCNU) Hodgkin's disease, non-Hodgkin'slymphomas, primary brain tumors, small-cell lung Semustine (methyl-CCNU)Primary brain tumors, stomach, colon Streptozocin Malignant pancreaticinsulinoma, (streptozotocin) malignant carcinoid Triazines Dacarbazine(DTIC; Malignant melanoma, Hodgkin's dimethyltriazenoimidaz disease,soft-tissue sarcomas olecarboxamide) Antimetabolites Folic Acid AnalogsMethotrexate Acute lymphocytic leukemia, (amethopterin) choriocarcinoma,mycosis fungoides, breast, head and neck, lung, osteogenic sarcomaPyrimidine Analogs Fluouracil (5-fluorouracil; Breast, colon, stomach,pancreas, 5-FU) ovary, head and neck, urinary bladder, Floxuridine(fluorode- premalignant skin lesions (topical) oxyuridine; FUdR)Cytarabine (cytosine Acute granulocytic and acute arabinoside)lymphocytic leukemias Purine Analogs and Mercaptopurine Acutelymphocytic, acute Related Inhibitors (6-mercaptopurine; granulocyticand chronic granulocytic 6-MP) leukemias Thioguanine Acute granulocytic,acute (6-thioguanine; TG) lymphocytic and chronic granulocytic leukemiasPentostatin Hairy cell leukemia, mycosis (2-deoxycoformycin) fungoides,chronic lymphocytic leukemia Natural Products Vinca AlkaloidsVinblastine (VLB) Hodgkin's disease, non-Hodgkin's lymphomas, breast,testis Vincristine Acute lymphocytic leukemia, neuroblastoma, Wilms'tumor, rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's lymphomas,small-cell lung Epipodophyllotoxins Etoposide Testis, small-cell lungand other lung, Tertiposide breast, Hodgkin's disease, non- Hodgkin'slymphomas, acute granulocytic leukemia, Kaposi's sarcoma AntibioticsDactinomycin Choriocarcinoma, Wilms' tumor, (actinomycin D)rhabdomyosarcoma, testis, Kaposi's sarcoma Daunorubicin Acutegranulocytic and acute (daunomycin; lymphocytic leukemias rubidomycin)Doxorubicin Soft-tissue, osteogenic and other sarcomas; Hodgkin'sdisease, non- Hodgkin's lymphomas, acute leukemias, breast,genitourinary, thyroid, lung, stomach, neuroblastoma Bleomycin Testis,head and neck, skin, esophagus, lung and genitourinary tract; Hodgkin'sdisease, non- Hodgkin's lymphomas Plicamycin (mithramycin) Testis,malignant hypercalcemia Mitomycin (mitomycin C) Stomach, cervix, colon,breast, pancreas, bladder, head and neck Enzymes L-Asparaginase Acutelymphocytic leukemia Biological Response Interferon alfa Hairy cellleukemia., Kaposi's Modifiers sarcoma, melanoma, carcinoid, renal cell,ovary, bladder, non-Hodgkin's lymphomas, mycosis fungoides, multiplemyeloma, chronic granulocytic leukemia Miscellaneous PlatinumCoordination Cisplatin (cis-DDP) Testis, ovary, bladder, head and neck,Agents Complexes Carboplatin lung, thyroid, cervix, endometrium,neuroblastoma, osteogenic sarcoma Anthracenedione Mitoxantrone Acutegranulocytic leukemia, breast Substituted Urea Hydroxyurea Chronicgranulocytic leukemia, polycythemia vera, essental thrombocytosis,malignant melanoma Methyl Hydrazine Procarbazine Hodgkin's diseaseDerivative (N-methylhydrazine, MIH) Adrenocortical Mitotane (o,p′-DDD)Adrenal cortex Suppressant Aminoglutethimide Breast Hormones andAdrenocorticosteroids Prednisone (several other Acute and chroniclymphocytic Antagonists equivalent leukemias, non-Hodgkin's lymphomas,preparations available) Hodgkin's disease, breast ProgestinsHydroxyprogesterone Endometrium, breast caproate Medroxyprogesteroneacetate Megestrol acetate Estrogens Diethylstilbestrol Breast, prostateEthinyl estradiol (other preparations available) Antiestrogen TamoxifenBreast Androgens Testosterone propionate Breast Fluoxymesterone (otherpreparations available) Antiandrogen Flutamide ProstateGonadotropin-releasing Leuprolide Prostate hormone analog

J2. Anti-Angiogenics

The term “angiogenesis” refers to the generation of new blood vessels,generally into a tissue or organ. Under normal physiological conditions,humans or animals undergo angiogenesis only in very specific restrictedsituations. For example, angiogenesis is normally observed in woundhealing, fetal and embryonic development and formation of the corpusluteum, endometrium and placenta. Uncontrolled (persistent and/orunregulated) angiogenesis is related to various disease states, andoccurs during tumor growth and metastasis.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

As persistent, unregulated angiogenesis occurs during tumor developmentand metastasis, the treatment methods of this invention may be used incombination with any one or more “anti-angiogenic” therapies. Exemplaryanti-angiogenic agents that are useful in connection with combinedtherapy are listed in Table D. Each of the agents listed therein isexemplary and by no means limiting.

TABLE D Inhibitors and Negative Regulators of Angiogenesis SubstancesReferences Angiostatin O'Reilly et al., 1994 Endostatin O'Reilly et al.,1997 16 kDa prolactin fragment Ferrara et al., 1991; Clapp et al., 1993;D'Angelo et al., 1995; Lee et al., 1998 Laminin peptides Kleinman etal., 1993; Yamamura et al., 1993; Iwamoto et al., 1996; Tryggvason, 1993Fibronectin peptides Grant et al., 1998; Sheu et al., 1997 Tissuemetalloproteinase inhibitors Sang, 1998 (TIMP 1, 2, 3, 4) Plasminogenactivator inhibitors Soff et al., 1995 (PAI-1, -2) Tumor necrosis factorα (high Frater-Schroder et al., 1987 dose, in vitro) TGF-β1 Ray Chadhuryand D'Amore, 1991; Tada et al., 1994 Interferons (IFN-α, -β, γ) Moore etal., 1998; Lingen et al., 1998 ELR-CXC Chemokines: Moore et al., 1998;Hiscox and Jiang, IL-12; SDF-1; MIG; Platelet factor 1997; Coughlin etal., 1998; Tanaka 4 (PF-4); IP-10 et al., 1997 Thrombospondin (TSP) Goodet al., 1990; Frazier, 1991; Bornstein, 1992; Tolsma et al., 1993;Sheibani and Frazier, 1995; Volpert et al., 1998 SPARC Hasselaar andSage, 1992; Lane et al., 1992; Jendraschak and Sage, 19962-Methoxyoestradiol Fotsis et al., 1994 Proliferin-related proteinJackson et al., 1994 Suramin Gagliardi et al., 1992; Takano et al.,1994; Waltenberger et al., 1996; Gagliardi et al., 1998; Manetti et al.,1998 Thalidomide D'Amato et al., 1994; Kenyon et al., 1997 Wells, 1998Cortisone Thorpe et al., 1993 Folkman et al., 1983 Sakamoto et al., 1986Linomide Vukanovic et al., 1993; Ziche et al., 1998; Nagler et al., 1998Fumagillin (AGM-1470; TNP-470) Sipos et al., 1994; Yoshida et al., 1998Tamoxifen Gagliardi and Collins, 1993; Linder and Borden, 1997; Haran etal., 1994 Korean mistletoe extract Yoon et al., 1995 (Viscum albumcoloratum) Retinoids Oikawa et al., 1989; Lingen et al., 1996; Majewskiet al. 1996 CM101 Hellerqvist et al., 1993; Quinn et al., 1995; Wamil etal., 1997; DeVore et al., 1997 Dexamethasone Hori et al., 1996; Wolff etal., 1997 Leukemia inhibitory factor (LIF) Pepper et al., 1995

A certain preferred component for use in inhibiting angiogenesis is aprotein named “angiostatin”. This component is disclosed in U.S. Pat.Nos. 5,776,704; 5,639,725 and 5,733,876, each incorporated herein byreference. Angiostatin is a protein having a molecular weight of betweenabout 38 kD and about 45 kD, as determined by reducing polyacrylamidegel electrophoresis, which contains approximately Kringle regions 1through 4 of a plasminogen molecule. Angiostatin generally has an aminoacid sequence substantially similar to that of a fragment of murineplasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule.

The amino acid sequence of angiostatin varies slightly between species.For example, in human angiostatin, the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, human plasminogen may be used,as it has similar anti-angiogenic activity, as shown in a mouse tumormodel.

Certain anti-angiogenic therapies have already been shown to cause tumorregressions, and angiostatin is one such agent. Endostatin, a 20 kDaCOOH-terminal fragment of collagen XVIII, the bacterial polysaccharideCM101, and the antibody LM609 also have angiostatic activity. However,in light of their other properties, they are referred to asanti-vascular therapies or tumor vessel toxins, as they not only inhibitangiogenesis but also initiate the destruction of tumor vessels throughmostly undefined mechanisms. Their combination with the presentinvention is clearly envisioned.

Angiostatin and endostatin have become the focus of intense study, asthey are the first angiogenesis inhibitors that have demonstrated theability to not only inhibit tumor growth but also cause tumorregressions in mice. There are multiple proteases that have been shownto produce angiostatin from plasminogen including elastase, macrophagemetalloelastase (MME), matrilysin (MMP-7), and 92 kDa gelatinase B/typeIV collagenase (MMP-9).

MME can produce angiostatin from plasminogen in tumors andgranulocyte-macrophage colony-stimulating factor (GMCSF) upregulates theexpression of MME by macrophages inducing the production of angiostatin.The role of MME in angiostatin generation is supported by the findingthat MME is in fact expressed in clinical samples of hepatocellularcarcinomas from patients. Another protease thought to be capable ofproducing angiostatin is stromelysin-1 (MMP-3). MMP-3 has been shown toproduce angiostatin-like fragments from plasminogen in vitro.

The mechanism of action for angiostatin is currently unclear, it ishypothesized that it binds to an unidentified cell surface receptor onendothelial cells inducing endothelial cell to undergo programmed celldeath or mitotic arrest. Endostatin appears to be an even more powerfulanti-angiogenesis and anti-tumor agent although its biology is much lessclear. Endostatin is effective at causing regressions in a number oftumor models in mice. Tumors do not develop resistance to endostatinand, after multiple cycles of treatment, tumors enter a dormant stateduring which they do not increase in volume. In this dormant state, thepercentage of tumor cells undergoing apoptosis was increased, yielding apopulation that essentially stays the same size. Endostatin is alsothought to bind an unidentified endothelial cell surface receptor thatmediates its effect.

CM101 is a bacterial polysaccharide that has been well characterized inits ability to induce neovascular inflammation in tumors. CM101 binds toand cross-links receptors expressed on dedifferentiated endothelium thatstimulates the activation of the complement system. It also initiates acytokine-driven inflammatory response that selectively targets thetumor. It is a uniquely antipathoangiogenic agent that downregulates theexpression VEGF and its receptors. CM101 is currently in clinical trialsas an anti-cancer drug, and can be used in combination herewith.

Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also be used incombination with the present invention. These are both angiogenesisinhibitors that associate with heparin and are found in plateletα-granules. TSP-1 is a large 450 kDa multi-domain glycoprotein that isconstituent of the extracellular matrix. TSP-1 binds to many of theproteoglycan molecules found in the extracellular matrix including,HSPGs, fibronectin, laminin, and different types of collagen. TSP-1inhibits endothelial cell migration and proliferation in vitro andangiogenesis in vivo. TSP-1 can also suppress the malignant phenotypeand tumorigenesis of transformed endothelial cells. The tumor suppressorgene p53 has been shown to directly regulate the expression of TSP-1such that, loss of p53 activity causes a dramatic reduction in TSP-1production and a concomitant increase in tumor initiated angiogenesis.

PF4 is a 70aa protein that is member of the CXC ELR- family ofchemokines that is able to potently inhibit endothelial cellproliferation in vitro and angiogenesis in vivo. PF4 administeredintratumorally or delivered by an adenoviral vector is able to cause aninhibition of tumor growth.

Interferons and metalloproteinase inhibitors are two other classes ofnaturally occurring angiogenic inhibitors that can be combined with thepresent invention. The anti-endothelial activity of the interferons hasbeen known since the early 1980s, however, the mechanism of inhibitionis still unclear. It is known that they can inhibit endothelial cellmigration and that they do have some anti-angiogenic activity in vivothat is possibly mediated by an ability to inhibit the production ofangiogenic promoters by tumor cells. Vascular tumors in particular aresensitive to interferon, for example, proliferating hemangiomas can besuccessfully treated with IFNα.

Tissue inhibitors of metalloproteinases (TIMPs) are a family ofnaturally occurring inhibitors of matrix metalloproteases (MMPs) thatcan also inhibit angiogenesis and can be used in combined treatmentprotocols with the present invention. MMPs play a key role in theangiogenic process as they degrade the matrix through which endothelialcells and fibroblasts migrate when extending or remodeling the vascularnetwork. In fact, one member of the MMPs, MMP-2, has been shown toassociate with activated endothelium through the integrin α_(v)β₃presumably for this purpose. If this interaction is disrupted by afragment of MMP-2, then angiogenesis is downregulated and in tumorsgrowth is inhibited.

There are a number of pharmacological agents that inhibit angiogenesis,any one or more of which may be used in combination with the presentinvention. These include AGM-1470/TNP-470, thalidomide, andcarboxyamidotriazole (CAI). Fumagillin was found to be a potentinhibitor of angiogenesis in 1990, and since then the syntheticanalogues of fumagillin, AGM-1470 and TNP-470 have been developed. Bothof these drugs inhibit endothelial cell proliferation in vitro andangiogenesis in vivo. TNP-470 has been studied extensively in humanclinical trials with data suggesting that long-term administration isoptimal.

Thalidomide was originally used as a sedative but was found to be apotent teratogen and was discontinued. In 1994 it was found thatthalidomide is an angiogenesis inhibitor. Thalidomide is currently inclinical trials as an anti-cancer agent as well as a treatment ofvascular eye diseases.

CAI is a small molecular weight synthetic inhibitor of angiogenesis thatacts as a calcium channel blocker that prevents actin reorganization,endothelial cell migration and spreading on collagen IV. CAI inhibitsneovascularization at physiological attainable concentrations and iswell tolerated orally by cancer patients. Clinical trials with CAI haveyielded disease stabilization in 49% of cancer patients havingprogressive disease before treatment.

Cortisone in the presence of heparin or heparin fragments was shown toinhibit tumor growth in mice by blocking endothelial cell proliferation.The mechanism involved in the additive inhibitory effect of the steroidand heparin is unclear although it is thought that the heparin mayincrease the uptake of the steroid by endothelial cells. The mixture hasbeen shown to increase the dissolution of the basement membraneunderneath newly formed capillaries and this is also a possibleexplanation for the additive angiostatic effect. Heparin-cortisolconjugates also have potent angiostatic and anti-tumor effects activityin vivo.

Further specific angiogenesis inhibitors, including, but not limited to,Anti-Invasive Factor, retinoic acids and paclitaxel (U.S. Pat. No.5,716,981; incorporated herein by reference); AGM-1470 (Ingber et al.,1990; incorporated herein by reference); shark cartilage extract (U.S.Pat. No. 5,618,925; incorporated herein by reference); anionic polyamideor polyurea oligomers (U.S. Pat. No. 5,593,664; incorporated herein byreference); oxindole derivatives (U.S. Pat. No. 5,576,330; incorporatedherein by reference); estradiol derivatives (U.S. Pat. No. 5,504,074;incorporated herein by reference); and thiazolopyrimidine derivatives(U.S. Pat. No. 5,599,813; incorporated herein by reference) are alsocontemplated for use as anti-angiogenic compositions for the combineduses of the present invention.

Compositions comprising an antagonist of an α_(v)β₃ integrin may also beused to inhibit angiogenesis in combination with the present invention.As disclosed in U.S. Pat. No. 5,766,591 (incorporated herein byreference), RGD-containing polypeptides and salts thereof, includingcyclic polypeptides, are suitable examples of α_(v)β₃ integrinantagonists.

The antibody LM609 against the α_(v)β₃ integrin also induces tumorregressions. Integrin α_(v)β₃ antagonists, such as LM609, induceapoptosis of angiogenic endothelial cells leaving the quiescent bloodvessels unaffected. LM609 or other α_(v)β₃ antagonists may also work byinhibiting the interaction of α_(v)β₃ and MMP-2, a proteolytic enzymethought to play an important role in migration of endothelial cells andfibroblasts.

Apoptosis of the angiogenic endothelium in this case may have a cascadeeffect on the rest of the vascular network. Inhibiting the tumorvascular network from completely responding to the tumor's signal toexpand may, in fact, initiate the partial or full collapse of thenetwork resulting in tumor cell death and loss of tumor volume. It ispossible that endostatin and angiostatin function in a similar fashion.The fact that LM609 does not affect quiescent vessels but is able tocause tumor regressions suggests strongly that not all blood vessels ina tumor need to be targeted for treatment in order to obtain ananti-tumor effect.

Non-targeted angiopoietins, such as angiopoietin-2, may also be used incombination with the present invention. As described above in thecontext of targeted delivery, the angiogenic effects of variousregulators involve an autocrine loop connected with angiopoietin-2. Theuse of angiopoietin-2, angiopoietin-1, angiopoietin-3 andangiopoietin-4, is thus contemplated in conjunction with the presentinvention. Other methods of therapeutic intervention based upon alteringsignaling through the Tie2 receptor can also be used in combinationherewith, such as using a soluble Tie2 receptor capable of blocking Tie2activation (Lin et al., 1998). Delivery of such a construct usingrecombinant adenoviral gene therapy has been shown to be effective intreating cancer and reducing metastases (Lin et al., 1998).

J3. Apoptosis-Inducing Agents

Therapeutic agent-targeting agent treatment may also be combined withtreatment methods that induce apoptosis in any cells within the tumor,including tumor cells and tumor vascular endothelial cells. Althoughmany anti-cancer agents may have, as part of their mechanism of action,an apoptosis-inducing effect, certain agents have been discovered,designed or selected with this as a primary mechanism, as describedbelow.

A number of oncogenes have been described that inhibit apoptosis, orprogrammed cell death. Exemplary oncogenes in this category include, butare not limited to, bcr-abl, bcl-2 (distinct from bcl-1, cyclin D1;GenBank accession numbers M14745, X06487; U.S. Pat. Nos. 5,650,491; and5,539,094; each incorporated herein by reference) and family membersincluding Bcl-xl, Mcl-1, Bak, A1, A20. Overexpression of bcl-2 was firstdiscovered in T cell lymphomas. bcl-2 functions as an oncogene bybinding and inactivating Bax, a protein in the apoptotic pathway.Inhibition of bcl-2 function prevents inactivation of Bax, and allowsthe apoptotic pathway to proceed. Thus, inhibition of this class ofoncogenes, e.g., using antisense nucleotide sequences, is contemplatedfor use in the present invention in aspects wherein enhancement ofapoptosis is desired (U.S. Pat. Nos. 5,650,491; 5,539,094; and5,583,034; each incorporated herein by reference).

Many forms of cancer have reports of mutations in tumor suppressorgenes, such as p53. Inactivation of p53 results in a failure to promoteapoptosis. With this failure, cancer cells progress in tumorigenesis,rather than become destined for cell death. Thus, provision of tumorsuppressors is also contemplated for use in the present invention tostimulate cell death. Exemplary tumor suppressors include, but are notlimited to, p53, Retinoblastoma gene (Rb), Wilm's tumor (WT1), baxalpha, interleukin-1b-converting enzyme and family, MEN-1 gene,neurofibromatosis, type 1 (NF1), cdk inhibitor p16, colorectal cancergene (DCC), familial adenomatosis polyposis gene (FAP), multiple tumorsuppressor gene (MTS-1), BRCA1 and BRCA2.

Preferred for use are the p53 (U.S. Pat. Nos. 5,747,469; 5,677,178; and5,756,455; each incorporated herein by reference), Retinoblastoma, BRCA1(U.S. Pat. No. 5,750,400; 5,654,155; 5,710,001; 5,756,294; 5,709,999;5,693,473; 5,753;441; 5,622,829; and 5,747,282; each incorporated hereinby reference), MEN-1 (GenBank accession number U93236) and adenovirusE1A (U.S. Pat. No. 5,776,743; incorporated herein by reference) genes.

Other compositions that may be used include genes encoding the tumornecrosis factor related apoptosis inducing ligand termed TRAIL, and theTRAIL polypeptide (U.S. Pat. No. 5,763,223; incorporated herein byreference); the 24 kD apoptosis-associated protease of U.S. Pat. No.5,605,826 (incorporated herein by reference); Fas-associated factor 1,FAFI (U.S. Pat. No. 5,750,653; incorporated herein by reference). Alsocontemplated for use in these aspects of the present invention is theprovision of interleukin-1β-converting enzyme and family members, whichare also reported to stimulate apoptosis.

Compounds such as carbostyril derivatives (U.S. Pat. Nos. 5,672,603; and5,464,833; each incorporated herein by reference); branched apogenicpeptides (U.S. Pat. No. 5,591,717; incorporated herein by reference);phosphotyrosine inhibitors and non-hydrolyzable phosphotyrosine analogs(U.S. Pat. Nos. 5,565,491; and 5,693,627; each incorporated herein byreference); agonists of RXR retinoid receptors (U.S. Pat. No. 5,399,586;incorporated herein by reference); and even antioxidants (U.S. Pat. No.5,571,523; incorporated herein by reference) may also be used. Tyrosinekinase inhibitors, such as genistein, may also be linked to ligands thattarget a cell surface receptor (U.S. Pat. No. 5,587,459; incorporatedherein by reference).

J4. Immunotoxins and Coaguligands

The anti-aminophospholipid-conjugate based treatment methods of theinvention may be used in combination with other immunotoxins and/orcoaguligands in which the targeting portion thereof, e.g., antibody orligand, is directed to a relatively specific marker of the tumor cells,tumor vasculature or tumor stroma. In common with the chemotherapeuticand anti-angiogenic agents discussed above, the combined use of othertargeted toxins or coagulants will generally result in additive,markedly greater than additive or even synergistic anti-tumor results.

Generally speaking, antibodies or ligands for use in these additionalaspects of the invention will preferably recognize accessible tumorantigens that are preferentially, or specifically, expressed in thetumor site. The antibodies or ligands will also preferably exhibitproperties of high affinity; and the antibodies, ligands or conjugatesthereof, will not exert significant in vivo side effects againstlife-sustaining normal tissues, such as one or more tissues selectedfrom heart, kidney, brain, liver, bone marrow, colon, breast, prostate,thyroid, gall bladder, lung, adrenals, muscle, nerve fibers, pancreas,skin, or other life-sustaining organ or tissue in the human body. Theterm “significant side effects”, as used herein, refers to an antibody,ligand or antibody conjugate, that, when administered in vivo, willproduce only negligible or clinically manageable side effects, such asthose normally encountered during chemotherapy.

At least one binding region of these second anti-cancer agents employedin combination with the invention will be a component that is capable ofdelivering a toxin or coagulation factor to the tumor region, i.e.,capable of localizing within a tumor site. Such targeting agents may bedirected against a component of a tumor cell, tumor vasculature or tumorstroma. The targeting agents will generally bind to a surface-expressed,surface-accessible or surface-localized component of a tumor cell, tumorvasculature or tumor stroma. However, once tumor vasculature and tumorcell destruction begins, internal components will be released, allowingadditional targeting of virtually any tumor component.

Many tumor cell antigens have been described, any one which could beemployed as a target in connection with the combined aspects of thepresent invention. Appropriate tumor cell antigens for additionalimmunotoxin and coaguligand targeting include those recognized by theantibodies B3 (U.S. Pat. No. 5,242,813; incorporated herein byreference; ATCC HB 10573); KSI/4 (U.S. Pat. No. 4,975,369; incorporatedherein by reference; obtained from a cell comprising the vectors NRRLB-18356 and/or NRRL B-18357); 260F9 (ATCC HB 8488); and D612 (U.S. Pat.No. 5,183,756; incorporated herein by reference; ATCC HB 9796). One mayalso consult the ATCC Catalogue of any subsequent year to identify otherappropriate cell lines producing anti-tumor cell antibodies.

For tumor vasculature targeting, the targeting antibody or ligand willoften bind to a marker expressed by, adsorbed to, induced on orotherwise localized to the intratumoral blood vessels of a vascularizedtumor. Appropriate expressed target molecules include, for example,endoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA (Liu et al.,1997), a TIE, a ligand reactive with LAM-1, a VEGF/VPF receptor, an FGFreceptor, α_(v)β₃ integrin, pleiotropin and endosialin. Suitableadsorbed targets are those such as VEGF, FGF, TGFβ, HGF, PF4, PDGF,TIMP, a ligand that binds to a TIE and tumor-associated fibronectinisoforms. Antigens naturally and artificially inducible by cytokines andcoagulants may also be targeted, such as ELAM-1, VCAM-1, ICAM-1, aligand reactive with LAM-1, endoglin, and even MHC Class II(cytokine-inducible, e.g., by IL-1, TNF-α, IFN-γ, IL4 and/or TNF-β); andE-selectin, P-selectin, PDGF and ICAM-1 (coagulant-inducible e.g., bythrombin, Factor IX/IXa, Factor X/Xa and/or plasmin).

The following patents and patent applications are specificallyincorporated herein by reference for the purposes of even furthersupplementing the present teachings regarding the preparation and use ofimmunotoxins directed against expressed, adsorbed, induced or localizedmarkers of tumor vasculature: U.S. Pat. Nos. 5,855,866; 5,776,427;5,863,538; 5,660,827; 5,877,289; 6,004,554; 5,965,132; 6,051,230; and6,093,399; and U.S. application Ser. No. 07/846,349.

Suitable tumor stromal targets include components of the tumorextracellular matrix or stroma, or components those bound therein;including basement membrane markers, type IV collagen, laminin, heparansulfate, proteoglycan, fibronectins, activated platelets, LIBS andtenascin. A preferred target for such uses is RIBS.

The following patents and patent applications are specificallyincorporated herein by reference for the purposes of even furthersupplementing the present teachings regarding the preparation and use oftumor stromal targeting agents: U.S. Pat. Nos. 5,877,289; 6,004,555;6,036,955; and 6,093,399.

The second anti-cancer therapeutics may be operatively attached to anyof the cytotoxic or otherwise anti-cellular agents described herein foruse in the anti-aminophospholipid immunotoxins. However, suitableanti-cellular agents also include radioisotopes. Toxin moieties will bepreferred, such as ricin A chain and deglycosylated A chain (dgA) oreven gelonin. Any one or more of the angiopoietins, or fusions thereof,may also be used as part of a second immunoconjugate for combinedtherapy.

The second, targeted agent for optional use with the invention maycomprise a targeted component that is capable of promoting coagulation,i.e., a coaguligand. Here, the targeting antibody or ligand may bedirectly or indirectly, e.g., via another antibody, linked to any factorthat directly or indirectly stimulates coagulation, including any ofthose described herein for use in the anti-aminophospholipidcoaguligands. Preferred coagulation factors for such uses are TissueFactor (TF) and TF derivatives, such as truncated TF (tTF), dimeric andmultimeric TF, and mutant TF deficient in the ability to activate FactorVII.

Effective doses of immunotoxins and coaguligands for combined use in thetreatment of cancer will be between about 0.1 mg/kg and about 2 mg/kg,and preferably, of between about 0.8 mg/kg and about 1.2 mg/kg, whenadministered via the IV route at a frequency of about 1 time per week.Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The physician responsible foradministration will determine the appropriate dose for the individualsubject.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I VCAM-1 Expression on Tumor and Normal Blood Vessels

A. Materials and Methods

1. Materials

Na¹²⁵I was obtained from Amersham (Arlington Heights, Ill.). Dulbecco'smodified Eagle's tissue culture medium (DMEM) and Dulbecco PBScontaining Ca²⁺ and Mg²⁺ were obtained from Gibco (Grand Island, N.Y.).Fetal calf serum was obtained from Hyclone (Logan, Utah).O-phenylenediamine, hydrogen peroxide, 3-aminopropyltriethoxy-silane andsterile, endotoxin-free saline (0.9% NaCl in 100 ml of water) were fromSigma (St. Louis, Mo.). SMPT was from Pierce (Rockford, Ill.). Proplex Tcontaining factor VII (74 IU/ml), factor X and factor IX (17 IU/ml) waspurchased from Baxter Diagnostics Inc. (McGraw Park, Ill.). Chromogenicsubstrate, S-2765, for measuring factor Xa proteolytic activity wasobtained from Chromogenix (Franklin, Ohio). Purified factor Xa waspurchased from American Diagnostica (Greenwich, Conn.). 96 and 48 flatbottom microtiter plates were obtained from Falcon (Becton Dickinson andCo., Lincoln Park, N.J.). Sepharose-Protein G beads and S200 Superdexwere purchased from Pharmacia (Piscataway, N.J.). Recombinant murineIL-1α was purchased from R&D Systems (Minneapolis, Minn.).

2. Antibodies

The MK2.7 hybridoma, secreting a rat IgG1 antibody against murineVCAM-1, was obtained from the American Type Culture Collection (ATCC,Rockville, Md.; ATCC CRL 1909). The characterization of this anti-VCAM-1antibody has been reported by Miyake et al. (1991, incorporated hereinby reference). The R187 hybridoma, secreting a rat IgG1 antibody againstmurine viral protein p30 gag, was also obtained from the ATCC, and wasused as an isotype matched control for the anti-VCAM-1 antibody.

Mouse monoclonal antibody, 10H10, against human tissue factor wasprepared as described in Morrissey et al. (1988), and in U.S.application Ser. No. 08/482,369, each incorporated herein by reference.

MECA 32, a pan anti-mouse vascular endothelial cell antibody, wasprepared as described by Leppink et al. (1989, incorporated herein byreference). MJ 7/18 rat IgG, reactive with murine endoglin, was preparedas described by Ge and Butcher (1994, incorporated herein by reference).The MECA 32 and MJ 7/18 antibodies served as positive controls forimmunohistochemical studies.

Rabbit anti-rat and rat anti-mouse secondary antibodies conjugated withhorseradish peroxidase (HRP) were purchased from Dako (Carpinteria,Calif.).

3. Antibody Purification

Anti-VCAM-1 hybridoma, MK 2.7, and the irrelevant control hybridoma,R187, were grown in bioreactors (Heraeus, Inc., Germany) for 12 days.Supernatants were centrifuged, filtered through 0.22 μm filters andloaded onto Sepharose-Protein G columns. IgG was eluted with citric acidbuffer, pH 3.5, dialyzed into PBS and stored thereafter at 4° C. in thesame buffer. Purity was estimated by SDS-PAGE and was routinely >90%.Binding capacity of the purified anti-VCAM-1 antibody was assessedimmunohistochemically on frozen sections of L540 tumor and by cell-basedELISA using IL-1α stimulated bEnd.3 cells, as described herein below.

4. Tumor-Bearing Mice and Immunohistochemistry

Male CB 17 SCID mice (Charles River, Wilmington, Mass.) weighingapproximately 25 g were injected with 1×10⁷ L540 Hodgkin's lymphomacells subcutaneously into the right flank. Tumors were allowed to growto a size of 0.4–0.7 cm³. Animals were anesthetized with metafane andtheir blood circulation was perfused with heparinized saline asdescribed by Burrows et al. (1992, incorporated herein by reference).The tumor and major organs were removed and snap-frozen in liquidnitrogen.

Cryostat sections of the tissues were cut, incubated with theanti-VCAM-1 antibody and stained immunohistochemically to detect VCAM-1.Rat IgG was detected using rabbit anti-rat IgG conjugated to HRPfollowed by development with carbazole (Fries et al., 1993).

B. Results

The blood vessels of the major organs and a tumor from mice bearingsubcutaneous L540 human Hodgkin's tumors were examinedimmunohistochemically for VCAM-1 expression using an anti-VCAM-1antibody. VCAM-1 expression on tumor blood vessels was more peripheralthan central. However, as demonstrated in Example VI and Example VII,the anti-VCAM-1 antibody and coaguligand were evidently binding to bloodtransporting vessels, as clearly shown by the ability of the coaguligandto arrest blood flow in all tumor regions and to cause destruction ofthe intratumoral region.

Overall, VCAM-1 expression was observed on 20–30% of total tumor bloodvessels stained by the anti-endoglin antibody, MJ 7/18. VCAM-1 stainingof the tumor vessels was largely observed on venules. VCAM-1 expressionwas similar in tumors up to 1500 mm³, but larger tumors appeared to havereduced staining, with 5–10% of MJ 7/18 positive vessels being positivefor VCAM-1.

Constitutive vascular expression of VCAM-1 was found in heart and lungsin both tumor-bearing and normal animals (Table 1). In the heart, strongstaining was observed on venules and veins. Approximately 10% of MECA 32positive vessels were positive for VCAM-1. Staining in lung endotheliumwas weak in comparison to heart and tumor, and was confined to a fewlarge blood vessels. Strong stromal staining was observed in testiswhere VCAM-1 expression was strictly extravascular. Similar findingsregarding constitutive VCAM-1 expression in rodent lung and testis werepreviously reported (Fries et al., 1993).

TABLE 1 Expression of VCAM-1 on Endothelium in Tissues of L540 TumorBearing Mice and Localization of Anti-VCAM-1 Antibody anti-VCAM-1 TissueVCAM-1 expression^(a) antibody localization^(b) Adrenal  −^(c) − BrainCerebellum − − Brain Cortex − − Heart ++ ++ Kidney − − Large Intestine −− Liver − − Lung + + Pancreas − − Small Intestine − − Spleen − − Testis −^(d) − L540 Hodgkin's tumor +++ +++ ^(a)VCAM-1 was detected byanti-VCAM-1 antibody followed by anti-rat IgG-HRP. ^(b)Localization ofanti-VCAM-1 antibody in vivo was determined by injecting the antibody,exsanguinating the mice and staining tissues staining with anti-ratIgG-HRP. ^(c)Intensity of staining was compared to pan-endothelialmarkers MJ 7/18 and MECA 32; − no staining; + weak; ++ moderate; +++strong. ^(d)No vascular expression was observed; however, extravascularstroma of testis was stained by anti-VCAM-1 antibody.

EXAMPLE II Localization of Anti-VCAM-1 Antibody In Vivo

A. Methods

Male CB 17 SCID mice (Charles River, Wilmington, Mass.) weighingapproximately 25 g were injected with 1×10⁷ L540 Hodgkin's lymphomacells subcutaneously into the right flank. Tumors were allowed to growto a size of 0.4–0.7 cm³.

Mice were injected intravenously with 30 μg/25 g body weight ofanti-VCAM-1 antibody, R187 antibody or corresponding coaguligands in 200μl of saline. Two hours later, animals were anesthetized with metafaneand their blood circulation was perfused with heparinized saline asdescribed (Burrows et al, 1992; incorporated herein by reference). Thetumor and major organs were removed and snap-frozen in liquid nitrogen.

Cryostat sections of the tissues were cut and were stainedimmunohistochemically for the presence of rat IgG or TF. Rat IgG wasdetected using rabbit anti-rat IgG conjugated to HRP followed bydevelopment with carbazole (Fries et al., 1993). Coaguligand wasdetected using the 10H10 antibody that recognizes human tissue factor,followed by HRP-labeled anti-mouse IgG. 10H10 antibody does notcross-react detectably with murine tissue factor (Morrissey et al, 1988,incorporated herein by reference) or other murine proteins.

B. Results

Mice bearing subcutaneous L540 tumors were injected intravenously withanti-VCAM-1 antibody and, two hours later, the mice were exsanguinated.The tumor and normal organs were removed and frozen sections wereprepared and examined immunohistochemically to determine the location ofthe antibody. Serial sections of the tissues were examined. Localizedrat IgG was detected by HRP-labeled anti-rat Ig; and murine bloodvessels were identified by pan-endothelial antibody, MECA 32.

Anti-VCAM-1 antibody was detected on endothelium of tumor, heart andlung (Table 1). The intensity and number of stained vessels wasidentical to that on serial sections of the same tissues staineddirectly with anti-VCAM-1 antibody (Table 1). Staining was specific asno staining of endothelium was observed in the tumor and organs of miceinjected with a species isotype matched antibody of irrelevantspecificity, R187. No localization of anti-VCAM-1 antibody was found intestis or any normal organ except heart and lung.

EXAMPLE III Preparation of Anti-VCAM-1∘tTF Coaguligand

An anti-VCAM-1∘tTF conjugate or “coaguligand” was prepared as follows.Truncated tissue factor (tTF), with an additional added cysteineintroduced at N-terminus (U.S. application Ser. No. 08/482,369,incorporated herein by reference), was expressed in E. coli and purifiedas described by Stone et al. (1995, incorporated herein by reference).After purification, the sulfhydryl group of N′ cysteine-tTF wasprotected by reaction with Ellman's reagent. The tTF derivative wasstored in small volumes at −70° C.

To prepare the anti-VCAM-1 coaguligand, 5 ml of anti-VCAM-1 antibody IgG(2 mg/ml) in PBS were mixed with 36 μl of SMPT (10 mM) dissolved in dryDMF and incubated at room temperature for 1 h. The mixture was filteredthrough a column of Sephadex G25 equilibrated in PBS containing 1 mMEDTA. The fractions containing the SMPT-derivatized antibody wereconcentrated to 4 ml by ultrafiltration in an Amicon cell equipped witha 10,000 Da cut-off filter. Freshly thawed tTF derivative was incubatedwith 30 μl of DTT (10 mM) in H₂O for 10 min. at room temperature and wasfiltered through a column of Sephadex G25 equilibrated in PBS containing1 mM EDTA. The eluted fractions containing reduced tTF were concentratedby ultrafiltration under nitrogen to a final volume of 3 ml.

The reduced tTF was mixed with the SMPT-derivatized antibody and themixture was allowed to react for 24 h at room temperature. At the end ofthe incubation, the reaction mixture was resolved by gel filtration on acolumn of Superdex S200 equilibrated in PBS. Fractions containinganti-VCAM-1∘tTF having a M_(r) of 180,000 and corresponding to onemolecule of antibody linked to one molecule of tTF were collected.

EXAMPLE IV Binding of Anti-VCAM-1 Coaguligand to Activated EndothelialCells

A. Methods

1. Iodination of 10H10 antibody

Anti-human tissue factor antibody, 10H10, was radiolabeled with ¹²⁵Iusing Chloramine T as described by Bocci (1964, incorporated herein byreference). The specific activity was approximately 10,000 cpm/μg, ascalculated from protein determinations measured by a Bradford assay(Bradford, 1976).

2. Cells

L540 Hodgkin cells (L540 Cy), derived from a patient with end-stagedisease, were prepared as described in Diehl et al. (1985, incorporatedherein by reference), and were obtained from Prof. Volker Diehl (Klinikfur Innere Medizin der Universitaet, Köeln, Germany). bEnd.3 cells(murine brain endothelioma) were prepared as described in Bussolino etal. (1991) and Montesano et al. (1990), each incorporated herein byreference, and were obtained from Prof. Werner Risau (Max PlanckInstitute, Bad Nauheim, Germany).

3. Tissue Culture

bEnd.3 cells and hybridomas were maintained in DMEM supplemented with10% fetal calf serum, 2 mM L-glutamine, 2 units/ml penicillin G and 2μg/ml streptomycin. L540 cells were maintained in RPMI 1640 containingthe same additives. All cells were subcultured once a week. bEnd.3trypsinization was performed using 0.125% trypsin in PBS solutioncontaining 0.2% EDTA. For binding studies, cells were seeded at adensity of 5×10⁴ cells/ml in 0.5 ml of medium in 48 well plates andincubated for 48–96 h. Medium was refreshed 24 h before each study.

4. Binding of Coaguligand to Activated Endothelial Cells

Binding of the anti-VCAM-1 antibody and coaguligand to VCAM-1 onactivated bEnd.3 cells was determined using a cell based ELISA, asdescribed by Hahne (1993, incorporated herein by reference). bEnd.3cells were incubated with 10 units/ml of IL-α for 4 h at 37° C. in96-well microtiter plates. At the end of this incubation, medium wasreplaced by DPBS containing 2 mM Ca²⁺ and Mg²⁺ and 0.2% (w/v) gelatin asa carrier protein. The same buffer was used for dilution of antibodiesand for washing of cell monolayers between steps.

Cells were incubated with 4 μg/ml of anti-VCAM-1•tTF conjugate,anti-VCAM•1 antibody or control reagents for 2 h, and were then washedand incubated for 1 h with rabbit anti-rat IgG-HRP conjugate (1:500dilution). All steps were performed at room temperature. HRP activitywas measured by adding O-phenylenediamine (0.5 mg/ml) and hydrogenperoxide (0.03% w/v) in citrate-phosphate buffer, pH 5.5. After 30 min.,100 μl of supernatant were transferred to 96 well plates, 100 μl of 0.18M H₂SO₄ were added and the absorbance was measured at 492 nm. Each studywas performed in duplicate and repeated at least twice.

5. Detection of Coaguligand Bound to Endothelial Cells

Anti-VCAM-1 coaguligand and appropriate controls were incubated withIL-1α stimulated bEnd.3 cells in 96-well microtiter plates, as describedabove. Bound coaguligands were detected by identifying both the tissuefactor component and the rat IgG component bound to bEnd.3 cells.

After removing the excess of unbound antibody, cells were incubated with100 μl/well of ¹²⁵I-labeled 10H10 antibody (0.2 μg/ml) or ¹²⁵I-labeledrabbit anti-rat Ig (0.2 μg/ml) in binding buffer. After 2 h incubationat room temperature, cells were washed extensively and dissolved in 0.5M of NaOH. The entire volume of 0.5 ml was transferred to plastic tubesand counted in a γ counter. Each study was performed in duplicate andrepeated at least twice.

B. Results

The ability of an anti-VCAM-1•tTF coaguligand to bind to IL-1α activatedmurine bEnd.3 cells was determined by measuring the binding ofradioiodinated anti-TF antibody to coaguligand-treated cells in vitro.VCAM-1 expression by bEnd.3 cells is transiently inducible by IL-1α witha peak of VCAM-1 expression being obtained 4–6 h after addition of thecytokine (Hahne et al., 1993). Strong binding of the coaguligand toactivated bEnd.3 cells was observed (FIG. 1A).

At saturation, 8.7 fmoles of anti-TF antibody was bound to the cells,which is equivalent to 540,000 molecules of anti-TF antibody per cell.Binding of the coaguligand was specific; no detectable binding overbackground was observed with an isotype matched control coaguligand ofirrelevant specificity. Binding of coaguligand to unstimulated cells wasabout half of that to activated cells and is probably attributable toconstitutive VCAM-1 expression by cultured endothelioma cells.

In further studies, the anti-VCAM-1•tTF coaguligand was found to bind asstrongly as unconjugated anti-VCAM-1 antibody to activated bEnd.3 cells,using detection by peroxidase-labeled anti rat IgG in the assay. Thiswas done at both saturating and subsaturating concentrations. Thus, theconjugation procedure (Example III) did not diminish antibody's capacityto bind to VCAM-1 on intact endothelial monolayers.

EXAMPLE V Factor X Activation by Endothelial Cell-Bound Coaguligand

A. Methods

The activity of the anti-VCAM-1-tTF coaguligand bound to activatedbEnd.3 cells was determined indirectly by using a chromogenic assay todetect factor Xa (Schorer et al., 1985; Nawroth et al., 1985; eachincorporated herein by reference). IL-1αα-stimulated and unstimulatedbEnd.3 cells were incubated with specific and control coaguligands in96-well microtiter plates as described above. The cells were washed withPBS containing 2 mg/ml of BSA and were incubated with 150 μl/well offreshly prepared Proplex T solution diluted 1:20 in 50 mM Tris-HC1 (pH8.1), 150 mM NaCl, 2 mg/ml BSA (tissue culture grade, endotoxin-free)and 2.5 mM CaCl₂. After incubation for 60 min. at 37° C., 100 μl werewithdrawn from each well, transferred to 96-well plates and mixed with10011 of the same buffer containing 12.5 mM EDTA (pH 8.1).

Chromogenic substrate S2765 for measuring factor Xa proteolytic activitywas added in 50 μl, giving a final concentration of 300 μM. Thebreakdown of the substrate was determined by reading the absorbance at405 nm over a 2 h period in a microplate reader (Molecular Devices, PaloAlto, Calif.).

Production of the chromogenic product was completely dependent on thepresence of Proplex T and bEnd.3 cells preincubated with the specificcoaguligand. Background hydrolysis of the substrate by Proplex T in theabsence of cells was approximately 10% of the maximal value and wassubtracted from each determination. Free coaguligands diluted in ProplexT solution were unable to generate factor Xa. The amount of Xa generatedwas calculated by reference to a standard curve constructed with knownconcentrations of purified factor Xa.

At the end of the study, cells were detached with trypsin-EDTA andcounted. The results are expressed as the amount of factor Xa generatedper 10⁴ cells. Each study was performed in duplicate and was repeated atleast 3 times.

B. Results

1 Factor X Activation

Anti-VCAM-1•tTF coaguligand bound to IL-1α-activated bEnd.3 cells wascapable of specifically activating factor X. The rate of generation offactor Xa by anti-VCAM-1-•tTF coated cells was 3.2 ng per 10⁴ cells perhour, which is 7–10 fold higher than was observed with activated cellstreated with a control coaguligand of irrelevant specificity or with tTFalone (FIG. 1B). Anti-VCAM-1•tTF in the absence of cells hadundetectable factor X activating activity, confirming that cell bindingis essential for coaguligand activity.

Anti-VCAM-1•tTF bound to unstimulated bEnd.3 cells activated factor X ata rate of 1.6 ng per 10⁴ cells per hour. This rate is about half thatobserved with the IL-1α-stimulated cells, in accordance with the 50%lower amount of coaguligand that binds to unstimulated as compared withstimulated cells. Similar results to those shown in FIG. 1B wereobtained in three separate studies.

2. Effect of Endothelial Cell Permeabilization

Permeabilization of bEnd.3 monolayers with saponin after treating themwith anti-VCAM-1•tTF coaguligand increased the ability of the boundcoaguligand to activate factor X by about 30-fold (Table 2). The rate offactor Xa generation by unstimulated cells treated with anti-VCAM-1•tTFincreased from 1.6 to 49.2 ng per 10⁴ cells per hour afterpermeabilization, while that of IL-1α stimulated cells increased from3.2 to 98.8 ng per 10⁴ cells per hour. The factor Xa generating activityof the permeabilized cells was due to the bound coaguligand rather thanto endogenous TF since permeabilized untreated cells or cells treatedwith control coaguligand of irrelevant specificity had low factor Xagenerating activity (2 ng per 10⁴ cells per hour).

These results indicate that the coaguligand is able to function moreefficiently in the environment of a permeabilized cell. Possibly,permeabilization exposes negatively-charged phospholipids from withinthe cell that accelerate the formation of the coagulation-initiationcomplexes, or else prevents the inactivation of such complexes by TFPI.

TABLE 2 Generation of Factor Xa by Anti-VCAM-1.tTF Bound to Intact orPermeabilized bEnd.3 cells (ng per 10⁴ cells per 60 min.) Intact cellsPermeabilized cells^(b) Treatment^(a) Control IL-1α Control IL-1α Buffer0.25^(c) 0.43 0.45 2.0 tTF 0.26 0.42 0.39 2.1 Control IgG.tTF 0.26 0.430.41 2.1 Anti-VCAM-1.tTF 1.64 3.17 49.2 98.8 ^(a)IL-1α stimulated andunstimulated bEnd.3 cells were incubated with buffer alone or with 4μg/ml of tTF, control IgG.tTF or anti-VCAM-1.tTF followed by 60 min.incubation with Proplex T solution at 37° C. ^(b)Cells were treated with0.2% saponin 5 min. before addition of Proplex T. ^(c)Amount of factorXa was determined as described above. Results are expressed as ng offactor Xa generated per 10⁴ cells per 60 min. The arithmetic mean valuesfrom triplicate wells are shown. SE were less than 5 percent of the meanvalues.

EXAMPLE VI Tumor Blood Vessel Thrombosis by Anti-VCAM-1 Coaguligand

A. Methods

SCID mice bearing L540 tumors (0.4–0.7 cm³) were injected intravenouslywith 40 μg (total protein) of anti-VCAM-1•tTF or R187•tTF. This dosecorresponds to 32 μg of antibody and 8 μg of tTF. Other animals receivedequivalent quantities of free antibody, free tTF or a mixture of both.Animals were anesthetized 4 or 24 h later and their blood circulationswere perfused with heparinized saline. The tumor and major organs wereremoved and were fixed in formalin and paraffin-embedded or snap-frozenfor cryosectioning. Sections were cut through the center of the tissueor tumor. The number of thrombosed and non-thrombosed blood vessels in 5cross-sections were counted. The percentage of thrombosed vessels wascalculated.

B. Results

1. Thrombosis of Tumor Blood Vessels

This study shows that intravenous administration of the anti-VCAM-1•tTFcoaguligand induces selective thrombosis of tumor blood vessels, asopposed to vessels in normal tissues, in tumor-bearing mice.

The anti-VCAM-1•tTF coaguligand was administered to mice bearingsubcutaneous L540 tumors of 0.4 to 0.6 cm in diameter. Beforecoaguligand injection, tumors were healthy, having a uniform morphologylacking regions of necrosis. The tumors were well vascularized and had acomplete absence of spontaneously thrombosed vessels or hemorrhages.Within four hours of coaguligand injection, 40–70% of blood vessels werethrombosed, despite the initial staining of only 20–30% of tumor bloodvessels shown in Example I. The thrombosed vessels contained occlusiveplatelet aggregates, packed erythrocytes and fibrin. In several regions,the blood vessels had ruptured, spilling erythrocytes into the tumorinterstitium.

By 24 h after coaguligand injection, the blood vessels were stilloccluded and extensive hemorrhage had spread throughout the tumor. Tumorcells had separated from one another, had pyknotic nuclei and wereundergoing cytolysis. By 72 h, advanced necrosis was evident throughoutthe tumor. Necrosis was clearly present in the intratumoral region ofthe tumor, where VCAM-1 expression on the vessels was not originallyprominent. The coaguligand binding was evidently effective to curtailblood flow in all tumor regions, resulting in widespread tumordestruction. Furthermore, it is likely that the initialcoaguligand-induced thrombin deposition results in increased inductionof the VCAM-1 target antigen on central vessels, thus amplifyingtargeting and tumor destruction.

The thrombotic action of anti-VCAM-1•tTF on tumor vessels was antigenspecific. None of the control reagents administered at equivalentquantities (tTF alone, anti-VCAM-1 antibody alone, tTF plus anti-VCAM-1antibody or the control coaguligand of irrelevant specificity) causedthrombosis (Table 3).

TABLE 3 Anti-VCAM-1.tTF-Mediated Thrombosis in L540 Tumor Bearing MiceThrombosed Vessels (%)^(b) Heart and Other Treatment^(a) L540 Tumor LungOrgans Saline 0–2 0 0 tTF 0–2 0 0 Anti-VCAM-1 Antibody 0–2 0 0Anti-VCAM-1 Antibody + tTF 0–2 0 0 Control IgG.tTF 0–2 0 0Anti-VCAM-1.tTF (<0.3 cm³)^(c)  0–10 0 0 Anti-VCAM-1.tTF (>0.3 cm³)40–70 0 0 ^(a)L540 tumor-bearing mice were injected i.v. with one of thefollowing reagents: saline; 8 μg of unconjugated tTF; 32 μg ofunconjugated anti-VCAM-1 antibody; mixture of 8 μg of tTF and 32 μg ofanti-VCAM-1 antibody; 40 μg of control IgG.tTF coaguligand; or 40 μg ofanti-VCAM-1.tTF coaguligand. Animals were sacrificed 4 h afterinjection. Tissues were removed and fixed in formalin. ^(b)Histologicalquantification was performed by counting numbers of thrombosed bloodvessels in 5 cross sections of tissue. The number of thrombosed vesselsis expressed as a percentage of total vessels. The range of results fromthree mice is given. ^(c)L540 tumor bearing mice were divided into twogroups (5–8 animals per group) having tumors smaller or larger than 0.3cm³.2. Lack of Thrombosis of Normal Blood Vessels

In addition to the thrombosis of tumor blood vessels, this study alsoshows that intravenous administration of the anti-VCAM-1•tTF coaguliganddoes not induce thrombosis of blood vessels in normal organs.

Despite expression of VCAM-1 on vessels in the heart and lung of normalor L540 tumor-bearing mice (Table 1), thrombosis did not occur afteranti-VCAM-1•tTF coaguligand administration. No signs of thrombosis,tissue damage or altered morphology were seen in mice injected with 5 to45 μg of coaguligand 4 or 24 h earlier. There was a normal histologicalappearance of the heart and lung from the same mouse that had majortumor thrombosis. All other major organs (brain, liver, kidney, spleen,pancreas, intestine, testis) also had unaltered morphology.

Frozen sections of organs and tumors from coaguligand-treated mice gavecoincident staining patterns when developed with either the anti-TFantibody, 10H10, or an anti-rat IgG antibody and confirmed that thecoaguligand had localized to vessels in the heart, lung and tumor. Theintensity of staining was equal to that seen when coaguligand wasapplied directly to the sections at high concentrations followed bydevelopment with anti-TF or anti-rat IgG, indicating that saturation ofbinding had been attained in vivo.

These studies show that binding of coaguligand to VCAM-1 on normalvasculature in heart and lung is not sufficient to induce thrombosis,and that tumor vasculature provides additional factors to supportcoagulation.

EXAMPLE VII In Vivo Tumor Destruction by Anti-VCAM-1 Coaguligand

A. Methods

Male CB17 SCID mice were injected subcutaneously with 1×10⁷ L540 cellsas described above. When the tumors had reached a volume of 0.4–0.6 cm³,the mice were injected intravenously with either 20 μg ofanti-VCAM-1-tTF, 16 μg anti-VCAM-1 antibody, 4 μg tTF, a mixture of 16μg of anti-VCAM-1 antibody and 4 μg of tTF, 20 μg control IgG•tTF orsaline. In some studies, the treatment was given 3 times, on days 0, 4and 8. A minimum of 8 animals were treated in each group.

Animals were monitored daily for tumor measurements and body weight.Mice were sacrificed when tumors had reached a diameter of 2 cm³, orearlier if tumors showed signs of necrosis or ulceration. Tumor volumewas calculated according to the formula: π/6×D×d², where D is the largertumor diameter and d is the smaller diameter. Differences in tumorgrowth rates were tested for statistical significance using anon-parametric test (Mann-Whitney rank sum test) that makes noassumptions about tumor size being normally distributed (Gibbons, 1976).

B. Results

The anti-tumor activity of anti-VCAM-1•tTF coaguligand was determined inSCID mice bearing 0.3–0.4 cm³ L540 tumors. The drug was administeredi.v. 3 times at intervals of 4 days. The pooled results from 3 separatestudies are presented in FIG. 2 and Table 4. Mean tumor volume ofanti-VCAM-1•tTF treated mice was significantly reduced at 21 days oftreatment (P<0.001) in comparison to all other groups. Nine of a totalof 15 mice treated with the specific coaguligand showed more than 50%reduction in tumor volume. This effect was specific since unconjugatedtTF, control IgG coaguligand and mixture of free anti-VCAM-1 antibodyand tTF did not affect tumor growth.

TABLE 4 Inhibition of Tumor Growth by Anti-VCAM-1.tTF Coaguligand Meantumor volume Tumor (mm³)^(b) Growth P versus Treatment^(a) n Day 0 Day21 Index^(c) saline^(d) Saline 14 331 ± 61 2190 ± 210 6.91 — TTF 13 341± 22 2015 ± 205 5.90 NS Anti-VCAM-1 16 363 ± 24 1920 ± 272 5.28 NSAnti-VCAM-1 + tTF 13 349 ± 42 2069 ± 362 5.92 NS Control IgG.tTF  8 324± 30 2324 ± 304 7.17 NS Anti-VCAM-1.tTF 15 365 ± 28 1280 ± 130 3.50<0.001 ^(a)L540 tumor bearing mice were injected i.v. with one of thefollowing reagents: saline; 8 μg of unconjugated tTF; 32 μg ofunconjugated anti-VCAM-1 antibody; mixture of 8 μg of tTF and 32 μg ofanti-VCAM-1 antibody; 40 μg of control IgG.tTF (R187) coaguligand; or 40μg of anti-VCAM-1.tTF coaguligand. The treatment was repeated on day 4and 7 after first injection. ^(b)Mean tumor volume ± SD. ^(c)The tumorgrowth index is the ratio of mean tumor volume on day 21 to mean tumorvolume on day 0. ^(d)Two tailed P values are for differences in tumorvolume (day 21) for the treated groups versus the saline group asdetermined by the Mann-Whitney rank sum test.

EXAMPLE VIII Phosphatidylserine Expression on Tumor Blood Vessels

A. Methods

1. Antibodies

Anti-phosphatidylserine (anti-PS) and anti-cardiolipin antibodies, bothmouse monoclonal IgM antibodies, were produced as described by Rote(Rote et al., 1993). Details of the characterization of the anti-PS andanti-cardiolipin antibodies were also reported by Rote et al. (1993,incorporated herein by reference).

2. Detection of PS Expression on Vascular Endothelium

L540 tumor-bearing mice were injected i.v. with 20 μg of either anti-PSor anti-cardiolipin mouse IgM antibodies. After 10 min., mice wereanesthetized and their blood circulations were perfused with heparinizedsaline. Tumors and normal tissues were removed and snap-frozen. Serialsections of organs and tumors were stained with either HRP-labeledanti-mouse IgM for detection of anti-PS antibody or with anti-VCAM-1antibody followed by HRP-labeled anti-rat Ig.

To preserve membrane phospholipids on frozen sections, the followingprotocol was developed. Animals were perfused with DPBS containing 2.5mM Ca²⁺. Tissues were mounted on 3-aminopropyltriethoxysilane-coatedslides and were stained within 24 h. No organic solvents, formaldehydeor detergents were used for fixation or washing of the slides. Slideswere re-hydrated by DPBS containing 2.5 mM Ca²⁺ and 0.2% gelatin. Thesame solution was also used to wash sections to remove the excess ofreagents. Sections were incubated with HRP-labeled anti-mouse IgM for3.5 h at room temperature to detect anti-PS IgM.

B. Results

To explain the lack of thrombotic effect of anti-VCAM-1•tTF on VCAM-1positive vasculature in heart and lungs, the inventors developed aconcept of differential PS localization between normal and tumor bloodvessels. Specifically, they hypothesized that endothelial cells innormal tissues segregate PS to the inner surface of the plasma membranephospholipid bilayer, where it is unable to participate in thromboticreactions; whereas endothelial cells in tumors translocate PS to theexternal surface of the plasma membrane, where it can support thecoagulation action of the coaguligand. PS expression on the cell surfaceallows coagulation because it enables the attachment of coagulationfactors to the membrane and coordinates the assembly of coagulationinitiation complexes (Ortel et al., 1992).

The inventors' model of PS translocation to the surface of tumor bloodvessel endothelial cells, as developed herein, is surprising in that PSexpression does not occur after, and does not inevitably trigger, celldeath. PS expression at the tumor endothelial cell surface is thussufficiently stable to allow PS to serve as a targetable entity fortherapeutic intervention.

To confirm the hypothesis that tumor blood vessel endothelium expressesPS on the luminal surface of the plasma membrane, the inventors usedimmunohistochemistry to determine the distribution of anti-PS antibodyafter intravenous injection into L540 tumor bearing mice. Anti-PSantibody localized within 10 min. to the majority of tumor bloodvessels, including vessels in the central region of the tumor that canlack VCAM-1. Vessels that were positive for VCAM-1 were also positivefor PS. Thus, there is coincident expression of PS on VCAM-1-expressingvessels in tumors.

In the in vivo localization studies, none of the vessels in normalorgans, including VCAM-1-positive vasculature of heart and lung, werestained, indicating that PS is absent from the external surface of theendothelial cells. In contrast, when sections of normal tissues andtumors were directly stained with anti-PS antibody in vitro, nodifferences were visible between normal and tumor, endothelial or othercell types, showing that PS is present within these cells but onlybecomes expressed on the surface of endothelial cells in tumors.

The specificity of PS detection was confirmed by two independentstudies. First, a mouse IgM monoclonal antibody directed against adifferent negatively charged lipid, cardiolipin, did not home to tumoror any organs in vivo. Second, pretreatment of frozen sections withacetone abolished staining with anti-PS antibody, presumably because itextracted the lipids together with the bound anti-PS antibody.

EXAMPLE IX Annexin V Blocks Coaguligand Activation of Factor X In Vitro

A. Methods

The ability of Annexin V to affect Factor Xa formation induced bycoaguligand was determined by a chromogenic assay described above inExample V. IL-1α-stimulated bEnd.3 cells were incubated withanti-VCAM-∘tTF and permeabilized by saponin. Annexin V was added atconcentrations ranging from 0.1 to 10 μg/ml and cells were incubated for30 min. before addition of diluted Proplex T. The amount of Factor Xagenerated in the presence or absence of Annexin V was determined asdescribed in Example V. Each treatment was performed in duplicate andrepeated at least twice.

B. Results

The need for surface PS expression in coaguligand action is furtherindicated by the inventors' finding that annexin V, which binds to PSwith high affinity, blocks the ability of anti-VCAM-1•tTF bound tobEnd.3 cells to generate factor Xa in vitro.

Annexin V added to permeabilized cells preincubated with anti-VCAM-1•tTFinhibited the formation of factor Xa in a dose-dependent manner (FIG.3). In the absence of Annexin V, cell-bound coaguligand produced 95 ngof factor Xa per 10,000 cells per 60 min. The addition of increasingamounts of Annexin V (in the μg per ml range) inhibited factor Xaproduction. At 10 μg per ml, Annexin V inhibited factor Xa production by58% (FIG. 3). No further inhibition was observed by increasing theconcentration of Annexin V during the assay, indicating that annexin Vsaturated all available binding sites at 10 μg per ml.

EXAMPLE X Annexin V Blocks Coaguligand Activity In Vivo

A. Methods

The ability of Annexin V to inhibit coaguligand-induced thrombosis invivo was examined in L540 Hodgkin-bearing SCID mice. Tumors were grownin mice as described above in Example II. Two mice per group (tumor size0.5 cm in diameter) were injected intravenously via the tail vein withone of the following reagents: a) saline; b) 100 μg of Annexin V; c) 40μg of anti-VCAM-1•tTF; d) 100 μg of Annexin V followed 2 hours later by40 μg of anti-VCAM-1•tTF.

Four hours after the last injection mice were anesthetized and perfusedwith heparinized saline. Tumors were removed, fixed with 4% formalin,paraffin-embedded and stained with hematoxylene-eosin. The number ofthrombosed and non-thrombosed blood vessels were counted and thepercentage of thrombosis was calculated.

B. Results

Annexin V also blocks the activity of the anti-VCAM-1∘tTF coaguligand invivo. Groups of tumor-bearing mice were treated with one of the controlor test reagents, as described in the methods. Mice were given (a)saline; (b) 100 μg of Annexin V; (c) 40 μg of anti-VCAM-1∘tTFcoaguligand; or (d) 100 μg of Annexin V followed 2 hours later by 40 μgof anti-VCAM-1∘tTF coaguligand. Identical results were obtained in bothmice per group.

No spontaneous thrombosis, hemorrhages or necrosis were observed intumors derived from saline-injected mice. Treatment with Annexin V alonedid not alter tumor morphology.

In accordance with other data presented herein, 40 μg of anti-VCAM-1∘tTFcoaguligand caused thrombosis in 70% of total tumor blood vessels. Themajority of blood vessels were occluded with packed erythrocytes andclots, and tumor cells were separated from one another. Bothcoaguligand-induced anti-tumor effects, i.e., intravascular thrombosisand changes in tumor cell morphology, were completely abolished bypre-treating the mice with Annexin V.

These findings confirm that the anti-tumor effects of coaguligands aremediated through the blockage of tumor vasculature. These data alsodemonstrate that PS is essential for coaguligand-induced thrombosis invivo.

EXAMPLE XI Externalized Phosphatidylserine is a Global Marker of TumorBlood Vessels

A. Methods

PS exposure on tumor and normal vascular endothelium was examined inthree animal tumor models: L540 Hodgkin lymphoma, NCI-H358 non-smallcell lung carcinoma, and HT 29 colon adenocarcinoma (ATCC). To grow thetumors in vivo, 2×10⁶ cells were injected into the right flank of SCIDmice and allowed to reach 0.8–1.2 cm in diameter. Mice bearing largetumors (volume above 800 mm³) were injected intravenously via the tailvein with 20 μg of either anti-PS or anti-cardiolipin antibodies. Theanti-cardiolipin antibody served as a control for all studies since bothantibodies are directed against negatively charged lipids and belong tothe same class of immunoglobulins (mouse IgM).

One hour after injection, mice were anesthetized and their bloodcirculation was perfused with heparinized saline. Tumors and normalorgans were removed and snap-frozen. Frozen sections were stained withanti-mouse IgM-peroxidase conjugate (Jackson Immunoresearch Labs)followed by development with carbazole.

B. Results

The anti-PS antibodies specifically homed to the vasculature of allthree tumors (HT 29, L540 and NCI-H358) in vivo, as indicated bydetection of the mouse IgM. The average percentages of vessels stainedin the tumors were 80% for HT 29, 30% for L540 and 50% for NCI-H358.Vessels in all regions of the tumors were stained and there was stainingboth of small capillaries and larger vessels.

No vessel staining was observed with anti-PS antibodies in any normaltissues. In the kidney, tubules were stained both with anti-PS andanti-CL, and this likely relates to the secretion of IgMs by this organ(Table 5). Anti-cardiolipin antibodies were not detected in any tumorsor normal tissues, except kidney.

These findings indicate that only tumor endothelium exposes PS to theouter site of the plasma membrane.

TABLE 5 Vessel Localization of Anti-PS and Anti-Cardiolipin Abs inTumor- Bearing Mice* Tissue Anti-PS† Anti-Cardiolipin† L540 Cy tumor ++− H358 tumor ++ − HT29 tumor +++ − Adrenal − − Brain Cerebellum − −Brain Cortex − − Heart − − Kidney  −‡  −‡ Large Intestine − − Liver − −Lung − − Pancreas − − Small Intestine − − Spleen − − Testes − −*Biodistribution in normal organs of both anti-PS and anti-cardiolipinAbs was identical in all three tumor animal models. †Anti-PS andanti-cardiolipin antibodies were detected on frozen sections usinganti-mouse IgM-peroxidase conjugate. − no staining; + weak; ++ moderate;+++ strong, equivalent to pan endothelial marker Meca 32. ‡Tubularstaining was observed in the kidneys of both and-PS and anti-CLrecipients.

To estimate the time at which tumor vasculature loses the ability tosegregate PS to the inner side of the membrane, the inventors examinedanti-PS localization in L540 tumors ranging in volume from 140 to 1,600mm³. Mice were divided into 3 groups according to their tumor size:140–300, 350–800 and 800–1,600 mm³. Anti-PS Ab was not detected in threemice bearing small L540 tumors (up to 300 mm³) Anti-PS Ab localized in 3animals of 5 in the group of intermediate size L540 tumors and in allmice (4 out of 4) bearing large L540 tumors (Table 6). Percent ofPS-positive blood vessels from total (identified by pan endothelialmarker Meca 32) was 10–20% in the L540 intermediate group and 20–40% inthe group of large L540 tumors (Table 6).

TABLE 6 PS Externalization Detected in Mid and Large Sized Tumors No.Positive Tumor Size (mm³) Tumors/Total* % PS-Positive Vessels/Total†350–800 3/5 10–20 850–1,600 4/4 20–40 *Mice bearing L540 Cy tumors weredivided into three groups according to tumor size. 20 μg of anti-PSantibodies were injected i.v. and allowed to circulate for 1 hour. Mouseantibodies were detected on frozen sections using anti-mouseIgM-peroxidase conjugate. †Total number of blood vessels was determinedusing pan-endothelial Ab Meca 32. PS-positive and Meca-positive vesselswere counted in 4 fields per cross section of tumor. Range of %PS-positive vessels within the same group is shown.

EXAMPLE XII Anti-Tumor Effects of Unconjugated Anti-PhosphatidylserineAntibodies

A. Methods

The effects of anti-PS antibodies were examined in syngeneic andxenogeneic tumor models. For the syngeneic model, 1×10⁷ cells of murinecolorectal carcinoma Colo 26 (obtained from Dr. Ian Hart, ICRF, London)were injected subcutaneously into the right flank of Balb/c mice. In thexenogeneic model, a human Hodgkin's lymphoma L540 xenograft wasestablished by injecting 1×10⁷ cells subcutaneously into the right flankof male CB17 SCID mice. Tumors were allowed to grow to a size of about0.6–0.9 cm³ before treatment.

Tumor-bearing mice (4 animals per group) were injected i.p. with 20 μgof naked anti-PS antibody (IgM), control mouse IgM or saline. Treatmentwas repeated 3 times with a 48 hour interval. Animals were monitoreddaily for tumor measurements and body weight. Tumor volume wascalculated as described in Example VII. Mice were sacrificed when tumorshad reached 2 cm³, or earlier if tumors showed signs of necrosis orulceration.

B. Results

The growth of both syngeneic and xenogeneic tumors was effectivelyinhibited by treatment with naked anti-PS antibodies (FIG. 4A and FIG.4B). Anti-PS antibodies caused tumor vascular injury, accompanied bythrombosis, and tumor necrosis. The presence of clots and disintegrationof tumor mass surrounding blocked blood vessels was evident.

Quantitatively, the naked anti-PS antibody treatment inhibited tumorgrowth by up to 60% of control tumor volume in mice bearing large Colo26 (FIG. 4A) and L540 (FIG. 4B) tumors. No retardation of tumor growthwas found in mice treated with saline or control IgM. No toxicity wasobserved in mice treated with anti-PS antibodies, with normal organspreserving unaltered morphology, indistinguishable from untreated orsaline-treated mice.

Tumor regression started 24 hours after the first treatment and tumorscontinue to decline in size for the next 6 days. This was observed inboth syngeneic and immunocompromised tumor models, indicating that theeffect was mediated by immune status-independent mechanism(s). Moreover,the decline in tumor burden was associated with the increase ofalertness and generally healthy appearance of the animals, compared tocontrol mice bearing tumors larger than 1500 mm³. Tumor re-growthoccurred 7–8 days after the first treatment.

The results obtained with anti-PS treatment of L540 tumors are furthercompelling for the following reasons. Notably, the tumor necrosisobserved in L540 tumor treatment occurred despite the fact that thepercentage of vessels that stained positive for PS in L540 tumors wasless than in HT 29 and NCI-H358 tumors. This implies that even morerapid necrosis would likely result when treating other tumor types.Furthermore, L540 tumors are generally chosen as an experimental modelbecause they provide clean histological sections and they are, in fact,known to be resistant to necrosis.

EXAMPLE XIII Anti-Tumor Effects of Annexin Conjugates

The surprising finding that aminophospholipids are stable markers oftumor vasculature also means that antibody-therapeutic agent constructscan be used in cancer treatment. In addition to using antibodies astargeting agents, the inventors reasoned that annexins, and otheraminophospholipid-binding proteins, could also be used to specificallydeliver therapeutic agents to tumor vasculature. The following datashows the anti-tumor effects that result from the in vivo administrationof annexin-TF constructs.

A. Methods

An annexin V-tTF conjugate was prepared and administered to nu/nu micewith solid tumors. The tumors were formed from human HT29 colorectalcarcinoma cells that formed tumors of at least about 1.2 cm³. Theannexin V-tTF coaguligand (10 μg) was administered intravenously andallowed to circulate for 24 hours. Saline-treated mice were separatelymaintained as control animals. After the one day treatment period, themice were sacrificed and exsanguinated and the tumors and major organswere harvested for analysis.

B. Results

The annexin V-tTF conjugate was found to induce specific tumor bloodvessel coagulation in HT29 tumor bearing mice. Approximately 55% of thetumor blood vessels in the annexin V-tTF conjugate treated animals werethrombosed following a single injection. In contrast, there was minimalevidence of thrombosis in the tumor vasculature of the control animals.

EXAMPLE XIV Phosphatidylserine Translocation in the Tumor Environment

The discovery of PS as an in vivo surface marker unique to tumorvascular endothelial cells prompted the inventors to further investigatethe effect of a tumor environment on PS translocation and outer membraneexpression. The present example shows that exposing endothelial cells invitro to certain conditions that mimic those in a tumor duplicates theobserved PS surface expression in intact, viable cells.

A. Methods

Mouse bEnd.3 endothelial cells were seeded at an initial density of50,000 cells/well. Twenty-fours later cells were incubated withincreasing concentrations of H₂O₂ (from 10 μM to 500 μM) for 1 hour at37° C. or left untreated. At the end of the incubation, cells werewashed 3 times with PBS containing 0.2% gelatin and fixed with 0.25%glutaraldehyde. Identical wells were either stained with anti-PS IgM ortrypsinized and evaluated for viability by the Trypan Blue exclusiontest. For the anti-PS staining, after blocking with 2% gelatin for 10min., cells were incubated with 2 μg/ml of anti-PS antibody, followed bydetection with anti-mouse IgM-HRP conjugate.

Wells seeded with mouse bEnd.3 endothelial cells were also incubatedwith different effectors and compared to control, untreated wells afterthe same period of incubation at 37° C. The panel of effectors testedincluded TNF, LPS, bFGF, IL-1α, IL-1β and thrombin. After incubation,cells were washed and fixed and were again either stained with anti-PSIgM or evaluated for viability using the Trypan Blue exclusion test, asdescribed above.

B. Results

1. PS Induction by H₂O₂

Exposing endothelial cells to H₂O₂ at concentrations higher than 100 μMcaused PS translocation in ˜90% cells. However, this was accompanied bydetachment of the cells from the substrate and cell viability decreasingto about 50–60%. The association of surface PS expression withdecreasing cell viability is understandable, although it is stillinteresting to note that ˜90% PS translocation is observed with only a50–60% decrease in cell viability.

Using concentrations of H₂O₂ lower than 100 μM resulted in significantPS expression without any appreciable reduction in cell viability. Forexample, PS was detected at the cell surface of about 50% of cells inall H₂O₂ treated wells using H₂O₂ at concentrations as low as 20 μM. Itis important to note that, under these low H₂O₂ concentrations, thecells remained firmly attached to the plastic and to each other, showedno morphological changes and had no signs of cytotoxicity. Detailedanalyses revealed essentially 100% cell—cell contact, retention ofproper cell shape and an intact cytoskeleton.

The 50% PS surface expression induced by low levels of H₂O₂ was thusobserved in cell populations in which cell viability was identical tothe control, untreated cells (i.e., 95%). The PS expression associatedwith high H₂O₂ concentrations was accompanied by cell damage, and thePS-positive cells exposed to over 100 μM H₂O₂ were detached, floatingand had disrupted cytoskeletons.

The maintenance of cell viability in the presence of low concentrationsH₂O₂ is consistent with data from other laboratories. For example,Schorer et al. (1985) showed that human umbilical vein endothelial cells(HUVEC) treated with 15 μM H₂O₂ averaged 90 to 95% viability (reportedas 5% to 10% injury), whilst those exposed to 1500 μM H₂O₂ were only0%–50% viable (50% to 100% injured).

The use of H₂O₂ to mimic the tumor environment in vitro is alsoappropriate in that the tumor environment is rich in inflammatory cells,such as macrophages, PMNs and granulocytes, which produce H₂O₂ and otherreactive oxygen species. Although never before connected with stabletumor vascular markers, inflammatory cells are known to mediateendothelial cell injury by mechanisms involving reactive oxygen speciesthat require the presence of H₂O₂ (Weiss et al., 1981; Yamada et al.,1981; Schorer et al., 1985). In fact, studies have shown thatstimulation of PMNs in vitro produces concentrations of H₂O₂ sufficientto cause sublethal endothelial cell injury without causing cell death(measured by chromium release assays) or cellular detachment; and thatthese H₂O₂ concentrations are attainable locally in vivo (Schorer etal., 1985).

The present in vitro translocation data correlates with the earlierresults showing that anti-PS antibodies localize specifically to tumorvascular endothelial cells in vivo, and do not bind to cells in normaltissues. The finding that in vivo-like concentrations of H₂O₂ induce PStranslocation to the endothelial cell surface without disrupting cellintegrity has important implications in addition to validating theoriginal in vivo data and the inventors' therapeutic approaches.

Human, bovine and murine endothelial cells are all known to bePS-negative under normal conditions. Any previously documented PSexpression has always been associated with cell damage and/or celldeath. This is simply not the case in the present studies, where normalviability is maintained. This shows that PS translocation in tumorvascular endothelium is mediated by biochemical mechanisms unconnectedto cell damage. This is believed to be the first demonstration of PSsurface expression in morphologically intact endothelial cells and thefirst indication that PS expression can be disconnected from theapoptosis pathway(s). Returning to the operability of the presentinvention, these observations again confirm that PS is a sustainable,rather than transient, marker of tumor blood vessels and a suitablecandidate for therapeutic intervention.

2. PS Expression does not Correlate with Cell Activation

The relevance of this in vitro data to the tumor environment is alsostrengthened by the fact that other, general cell activators are withouteffect on PS translocation in endothelial cells. For example, theinventors tested TNF in similarly controlled studies and found it unableto induce PS surface expression, despite the expected increases inE-selectin and VCAM expression. Likewise, LPS, bFGF, IL-1α and IL-1βwere all without effect on PS expression in appropriately controlledstudies.

3. PS Induction by Thrombin

In contrast to the lack of effects of other cell activators, thrombinwas observed to increase PS expression, although not to the same extentas H₂O₂. This data is also an integral part of the tumor-induction modelof PS expression developed by the present inventors (thrombin-induced PSsurface expression in normal tissues would also further coagulation asPS expression coordinates the assembly of coagulation initiationcomplexes (Ortel et al., 1992)).

The tumor environment is known to be prothrombotic, such that tumorvasculature is predisposed to coagulation (U.S. Pat. No. 5,877,289). Asthrombin is a product of the coagulation cascade, it is present in tumorvasculature. In fact, the presence of thrombin induces VCAM expression,contributing to the inventors' ability to exploit VCAM as a targetablemarker of tumor vasculature (U.S. Pat. Nos. 5,855,866; 5,877,289). Thepresent data showing that thrombin also induces PS expression is thusboth relevant to targeting aminophospholipids with naked antibodies andtherapeutic conjugates, and further explains the beneficial effects ofthe anti-VCAM coaguligand containing Tissue Factor (Example VII).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Abrams and Oldham, In: Monoclonal Antibody Therapy of Human Cancer,    Foon and Morgan (Eds.), Martinus Nijhoff Publishing, Boston, pp.    103–120, 1985.-   Anderson, Croyle, Lingrel, “Primary structure of a gene encoding rat    T-kininogen,” Gene, 81(1):119:28, 1989.-   Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,    1988.-   Asahara, Chen, Takahashi, Fujikawa, Kearney, Magner, Yancopoulos,    Isner, “Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2,    modulate VEGF-induced postnatal neovascularization” Circ. Res.,    83(3):233–40, 1998.-   Barbas, Kang, Lerner, Benkovic, “Assembly of combinatorial antibody    libraries on phage surfaces: the gene III site,” Proc. Natl. Acad.    Sci., USA, 88(18):7978–7982, 1991.-   Berard, Boffa, Karmochkine, Aillaud, Juhan-Vague, Frances, Cacoub,    Piette, Harle, “Plasma reactivity to hexagonal II phase    phosphatidylethanolamine is more frequently associated with lupus    anticoagulant than with antiphosphatidylethanolamine antibodies,” J.    Lab. Clin. Med., 122(5):601–605, 1993.-   Berman, Mellis, Pollock, Smith, Suh, Heinke, Kowal, Surti, Chess,    Cantor, et al., “Content and organization of the human Ig VH locus:    definition of three new VH families and linkage to the Ig CH locus,”    EMBO J., 7(3):727–738, 1988.-   Bernier and Jolles, “Purification and characterization of a basic 23    kDa cytosolic protein from bovine brain,” Biochim. Biophys. Acta,    790(2):174–181, 1984.-   Bernier, Tresca, Jolles, “Ligand-binding studies with a 23 kDa    protein purified from bovine brain cytosol,” Biochim. Biophys. Acta,    871(1):19–23, 1986.-   Bevers, Comburius, Zwaal, “The nature of the binding site for    prothrombinase at the platelet surface as revealed by lipolytic    enzymes,” Eur. J. Biochem., 122:81–85, 1982.-   Bevers, Comfurius, Zwaal, “Changes in membrane phospholipid    distribution during platelet activation,” Biochim. Biophys. Acta,    736:57–66, 1983.-   Bevers, Rosing, Zwaal, “Development of procoagulant binding sites on    the platelet surface,” Adv. Exp. Med. Biol., 192:359–371, 1985.-   Bevers, Galli, Barbui, Comfurius, Zwaal, “Lupus anticoagulant IgG's    (LA) are not directed to phospholipids only, but to a complex of    lipid-bound human prothrombin,” Thromb. Haemost., 66(6):629–632,    1991.-   Bevilacqua, “Endothelial-leukocyte adhesion molecules,” Ann. Rev.    Immunol., 11:767–804, 1993.-   Blankenberg, Katsikis, Tait, Davis, Naumovski, Ohtsuki, Kopiwoda,    Abrams, Darkes, Robbins, Maecker, Strauss, “In vivo detection and    imaging of phosphatidylserine expression during programmed cell    death,” Proc. Natl. Acad. Sci., USA, 95:6349–6354, 1998.-   Bocci, “Efficient labeling of serum proteins with 131I using    chloramine T,” Int. J. Appl. Radiat. Isot., 15:449–456, 1964.-   Bombeli, Karsan, Tait, Harlan, “Apoptotic vascular endothelial cells    become procoagulant,” Blood, 89(7):2429–2442, 1997.-   Bordron, Dueymes, Levy, Jamin, Leroy, Piette, Schoenfeld, Youinou,    “The binding of some human antiendothelial cell antibodies induces    endothelial cell apoptosis,” J. Clin. Invest., 101(10):2029–2035,    1998.-   Bornstein, “Thrombospondins: structure and regulation of    expression,” FASEB J, 6(14):3290–3299, 1992.-   Borrebaeck and Moller, “In vitro immunization. Effect of growth and    differentiation factors on antigen-specific B cell activation and    production of monoclonal antibodies to autologous antigens and weak    immunogens,” J. Immunol., 136(10):3710–3715, 1986.-   Bradford, “A rapid and sensitive method for the quantitation of    microgram quantities of protein utilizing the principle of    protein-dye binding,” Anal. Biochem., 72:248–254, 1976.-   Branch, Rote, Dostal, Scott, “Association of lupus anticoagulant    with antibody against phosphatidylserine,” Clin. Immun.    Immunopathol., 42:63–75, 1987.-   Brinkman, Mertens, Holthius, Zwart-Huinink, Grijm, Van Mourik, “The    activation of human blood coagulation factor X on the surface of    endothelial cells: a comparison with various vascular cells,    platelets and monocytes,” Br. J. Haematol., 87:332–342, 1994.-   Bruijn and Dinklo, “Distinct patterns of expression of intercellular    adhesion molecule-1, vascular cell adhesion molecule-1, and    endothelial-leukocyte adhesion molecule-1 in renal disease,” Lab.    Invest., 69:329–335, 1993.-   Burke et al., “Cloning of large segments of exogenous DNA into yeast    by means of artificial chromosome vectors”, Science, 236, 806–812,    1987.-   Burrows and Thorpe, “Eradication of large solid tumors in mice with    an immunotoxin directed against tumor vasculature,” Proc. Natl.    Acad. Sci. USA, 90:8996–9000, 1993-   Burrows, Watanabe, Thorpe, “A murine model for antibody-directed    targeting of vascular endothelial cells in solid tumors,” Cancer    Res., 52:5954–5962, 1992.-   Bussolino, deRossi, Sica, Colotta, Wang, Bocchietto, Martin, Padura,    Bosia, Dejana, Mantovani, “Murine endothelial cell lines transformed    by polyoma middle T oncogene as target for and producers of    cytokines,” J. Immunol., 147:2122–2129, 1991.-   Campbell, In: Monoclonal Antibody Technology, Laboratory Techniques    in Biochemistry and Molecular Biology, Vol. 13, Burden and Von    Knippenberg (Eds.), Elseview, Amsterdam, pp. 75–83, 1984.-   Carnemolla et al., “A tumor-associated fibronectin isoform generated    by alternative splicing of messenger RNA precursors,” J. Cell Biol.,    108:1139–1148, 1989.-   Chamley, McKay, Pattison, “Cofactor dependent and cofactor    independent anticardiolipin antibodies,” Thromb. Res.,    61(3):291–299, 1991.-   Clapp et al., “The 16-kilodalton N-terminal fragment of human    prolactin is a potent inhibitor of angiogenesis,” Endocrinology,    133(3): 1292–1299, 1993.-   Connor, Bucana, Fidler, Schroit, “Differentiation-dependent    expression of phosphatidylserine in mammalian plasma membranes:    quantitative assessment of outer-leaflet lipid by prothrombinase    complex formation,” Proc. Natl. Acad. Sci. USA, 86(9):3184–3188,    1989.-   Coughlin et al., “Interleukin-12 and interleukin-18 synergistically    induce murine tumor regression which involves inhibition of    angiogenesis,” J. Clin. Invest., 101(6):1441–1452, 1998.-   Dachary-Prigent, Toti, Satta, Pasquet, Uzan, Freyssinet,    “Physiopathological significance of catalytic phospholipids in the    generation of thrombin,” Seminars In Thrombosis and Hemostasis,    22:157–164, 1996.-   D'Amato et al., “Thalidomide is an inhibitor of angiogenesis,” Proc.    Natl. Acad. Sci. USA, 91(9):4082–4085, 1994.-   D'Angelo et al., “Activation of mitogen-activated protein kinases by    vascular endothelial growth factor and basic fibroblast growth    factor in capillary endothelial cells is inhibited by the    antiangiogenic factor 16-kDa N-terminal fragment of prolactin,”    Proc. Natl. Acad. Sci. USA, 92(14):6374–6378, 1995.-   Davis and Yancopoulos, “The angiopoietins: Yin and Yang in    angiogenesis”, Curr. Top. Microbiol. Immunol., 237:173–85, 1999.-   de Jong, Geldwerth, Kuypers, “Oxidative damage does not alter    membrane phospholipid asymmetry in human erythrocytes,” Am. Chem.    Soc., 1997.-   Denekamp, “Vascular attack as a therapeutic strategy for cancer,”    Cancer Metastasis Rev., 9:267–282, 1990.-   DeVore et al., “Phase I Study of the Antineovascularization Drug    CM101,” Clin. Cancer Res., 3(3):365–372, 1997.-   Diehl, Pfreundschuh, Fonatsch, Stein, Falk, Burrichter, Schaadt,    “Phenotypic genotypic analysis of Hodgkin's disease derived cell    lines: histopathological and clinical implications,” Cancer Surveys,    4:399–416, 1985.-   Donati, “Cancer and thrombosis: from Phlegmasia alba dolens to    transgenic mice,” Thromb. Haemost., 74:278–281, 1995.-   Drouvalakis and Buchanan, “Phospholipid specificity of autoimmune    and drug induced lupus anticoagulants; association of    phosphatidylethanolamine reactivity with thrombosis in autoimmune    disease,” J. Rheumatol., 25(2):290–295, 1998.-   Droz, Patey, Paraf, Chretien, Gogusev, “Composition of extracellular    matrix and distribution of cell adhesion molecules in renal cell    tumors,” Lab. Invest., 71:710–718, 1994.-   Dvorak, Nagy, Dvorak, “Structure of Solid Tumors and Their    Vasculature: Implications for Therapy with Monoclonal Antibodies,”    Cancer Cells, 3(3):77–85, 1991.-   Edgington, Mackman, Brand, Ruf, “The Structural Biology of    Expression and Function of Tissue Factor,” Thromb. Haemost.,    66(1):67–79, 1991.-   Ferrara, Clapp, Weiner, “The 16K fragment of prolactin specifically    inhibits basal or fibroblast growth factor stimulated growth of    capillary endothelial cells,” Endocrinology, 129(2):896–900, 1991.-   Flynn, Byrne, Baglin, Weissberg, Bennett, “Thrombin generation by    apoptotic vascular smooth muscle cells,” Blood, 89(12):4378–4384,    1997.-   Folkman et al., “Angiogenesis inhibition and tumor regression caused    by heparin or a heparin fragment in the presence of cortisone,”    Science, 221:719–725, 1983.-   Fotsis et al., “The endogenous oestrogen metabolite    2-methoxyoestradiol inhibits angiogenesis and suppresses tumour    growth,” Nature, 368(6468):237–239, 1994.-   Frater-Schroder et al., “Tumor necrosis factor type alpha, a potent    inhibitor of endothelial cell growth in vitro, is angiogenic in    vivo,” Proc. Natl. Acad. Sci. USA, 84(15):5277–5281, 1987.-   Frazier, “Thrombospondins,” Curr. Opin. Cell Biol., 3(5):792–799,    1991.-   Fries, Williams, Atkins, Newman, Lipscomb, Collins, “Expression of    VCAM-1 and E-selectin in an in vivo model of endothelial    activation,” Am. J. Pathol., 143:725–737, 1993.-   Gaffet, Bettache, Bienvenüe, “Transverse redistribution of    phospholipids during human platelet activation: evidence for a    vectorial outflux specific to aminophospholipids,” Biochem.,    34:6762–6769, 1995.-   Gagliardi and Collins, “Inhibition of angiogenesis by    antiestrogens,” Cancer Res., 53(3):533–535, 1993.-   Gagliardi, Hadd, Collins, “Inhibition of angiogenesis by suramin,”    Cancer Res., 52(18):5073–5075, 1992.-   Gagliardi et al., “Antiangiogenic and antiproliferative activity of    suramin analogues,” Cancer Chemother. Pharmacol., 41(2): 117–124,    1998.-   Galli, Comfurius, Maassen Hemker, de Baets, van Breda-Vriesman,    Barbui, Zwaal, Bevers, “Anticardiolipin antibodies (ACA) directed    not to cardiolipin but to a plasma protein cofactor,” Lancet,    335(8705):1544–1547, 1990.-   Galli, Barbui, Zwaal, Comfurius, Bevers, “Antiphospholipid    antibodies: involvement of protein cofactors,” Haematologica,    78(1):1–4, 1993.-   Ge and Butcher, “Cloning and expression of a cDNA encoding mouse    endoglin, an endothelial cell TGF-beta ligand,” Gene, 138:201–206,    1994.-   Gefter et al., “A simple method for polyethylene glycol-promoted    hybridization of mouse myeloma cells,” Somatic Cell Genet.,    3:231–236, 1977.-   Gems, Ferguson, Robertson, Nieves, Page, Blaxter, Maizels, “An    abundant, trans-spliced mRNA from Toxocara canis invective larvae    encodes a 26-kDa protein with homology to    phosphatidylethanolamine-binding proteins,” J. Biol. Chem., 270(31):    18517–18522, 1995.-   Gibbons, “Mann-Whitney-Wilcoxon test for two independent samples,”    In: Nonparametric methods for quantitative analysis, J. D. Gibbons    (ed.), Holt, Rinehart and Winston, New York, pp. 160, 1976.-   Glennie, et al., “Preparation and performance of bispecific F(ab′    gamma)2 antibody containing thioether-linked Fab′ gamma    fragments,” J. Immunol., 139:2367–2375, 1987.-   Goding, In: Monoclonal Antibodies: Principles and Practice, 2nd    Edition, Academic Press, Orlando, Fl., pp. 60–61, 65–66, 71–74,    1986.-   Good et al., “A tumor suppressor-dependent inhibitor of angiogenesis    is immunologically and functionally indistinguishable from a    fragment of thrombospondin,” Proc. Natl. Acad. Sci. USA,    87(17):6624–6628, 1990.-   Grant et al., “Fibronectin fragments modulate human retinal    capillary cell proliferation and migration,” Diabetes,    47(8):1335–1340, 1998.-   Hagemeier et al., “A Monoclonal Antibody Reacting with Endothelial    Cells of Budding Vessels in Tumors and Inflammatory Tissues, and    Non-Reactive with Normal Adult Tissues,” Int. J. Cancer, 38:481–488,    1986.-   Hahne, Jager, Isenmann, Hallmann, Vestweber, “Five tumor necrosis    factor-inducible cell adhesion mechanisms on the surface of mouse    endothelioma cells mediate the binding of leukocytes,” J. Cell    Biol., 121:655–664, 1993.-   Hampton, Vanags, Porn-Ares, Orrenius, “Involvement of extracellular    calcium in phosphatidylserine exposure during apoptosis,” FEBS    Lett., 399(3):277–282, 1996.-   Haran et al., “Tamoxifen enhances cell death in implanted MCF7    breast cancer by inhibiting endothelium growth,” Cancer Res.,    54(21):5511–5514, 1994.-   Hasselaar and Sage, “SPARC antagonizes the effect of basic    fibroblast growth factor on the migration of bovine aortic    endothelial cells,” J. Cell Biochem., 49(3):272–283, 1992.-   Hellerqvist et al., “Antitumor effects of GBS toxin: a    polysaccharide exotoxin from group B beta-hemolytic streptococcus,”    J Cancer Res. Clin. Oncol., 120(1–2):63–70, 1993.-   Hiscox and Jiang, “Interleukin-12, an emerging anti-tumour    cytokine,” In Vivo, 11(2):125–132, 1997.-   Holash et al., “Vessel Cooption, Regression, and Growth in Tumors    Mediated by Angiopoietins and VEGF”, Science, 284:1994–1998, 1999.-   Hori, Chae, Murakawa, Matoba, Fukushima, Okubo, Matsubara, “A human    cDNA sequence homologue of bovine phosphatidylethanolamine-binding    protein,” Gene, 140(2):293–294, 1994.-   Hori et al., “Differential effects of angiostatic steroids and    dexamethasone on angiogenesis and cytokine levels in rat sponge    implants,” Br. J. Pharmacol., 118(7):1584–1591, 1996.-   Huang, Molema, King, Watkins, Edgington, Thorpe, “Tumor infarction    in mice by antibody-directed targeting of tissue factor to tumor    vasculature,” Science, 275:547–550, 1997.-   Huse, Sastry, Iverson, Kang, Alting-Mees, Burton, Benkovic, Lerner,    Science, 246(4935):1275–1281, 1989.-   Igarashi, Umeda, Tokita, Soo Nam, Inoue, “Effective induction of    anti-phospholipid and anticoagulant antibodies in normal mouse,”    Thrombosis Res., 61:135–148, 1991.-   Ingber et al., “Angioinhibins: Synthetic analogues of fumagillin    which inhibit angiogenesis and suppress tumor growth,” Nature,    48:555–557, 1990.-   Iwamoto et al., “Inhibition of angiogenesis, tumour growth and    experimental metastasis of human fibrosarcoma cells HT1080 by a    multimeric form of the laminin sequence Tyr-Ile-Gly-Ser-Arg    (YIGSR),” Br. J. Cancer, 73(5):589–595, 1996.-   Jackson et al., “Stimulation and inhibition of angiogenesis by    placental proliferin and proliferin-related protein,” Science,    266(5190):1581–1584, 1994.-   Jamasbi, Wan, Stoner, “Epitope masking of rat esophageal carcinoma    tumor-associated antigen by certain coexisting glycolipid and    phospholipid molecules: a potential mechanism for tumor cell escape    from the host immune responses,” Cancer Immunol Immunother.,    38(2):99–106, 1994.-   Jendraschak and Sage, “Regulation of angiogenesis by SPARC and    angiostatin: implications for tumor cell biology,” Semin. Cancer    Biol., 7(3):139–146, 1996.-   Jones and Hall, “A 23 kDa protein from rat sperm plasma membranes    shows sequence similarity and phospholipid binding properties to a    bovine brain cytosolic protein,” Biochim. Biophys. Acta,    1080(1):78–82, 1991.-   Jones, Dear, Foote, Neuberger, Winter, Nature, 321(6069):522–525,    1986.-   Julien, Tournier, Tocanne, “Differences in the transbilayer and    lateral motions of fluorescent analogs of phosphatidylcholine and    phosphatidylethanolamine in the apical plasma membrane of bovine    aortic endothelial cells,” Exp. Cell. Res., 208(2):387–389, 1993.-   Julien, Toumier, Tocanne, “Basic fibroblast growth factor modulates    the aminophospholipid translocase activity present in the plasma    membrane of bovine aortic endothelial cells,” Eur. J. Biochem.,    230:287–297, 1995.-   Julien, Millot, Tocanne, Tournier,    “12-O-Tetradecanoylphorbol-13-Acetate inhibits aminophospholipid    translocase activity and modifies the lateral motions of fluorescent    phospholipid analogs in the plasma membrane of bovine aortic    endothelial cells,” Experimental Cell Res., 234:125–131, 1997.-   Kang, Barbas, Janda, Benkovic, Lerner, Proc. Natl. Acad. Sci.,    U.S.A, 88(10):4363–4366, 1991.-   Katsuragawa, Kanzaki, Inoue, Hirano, Mori, Rote, “Monoclonal    antibody against phosphatidylserine inhibits in vitro human    trophoblastic hormone production and invasion,” Biology of    Reproduction, 56:50–58, 1997.-   Kellermann, Lottspeich, Henschen, Muller-Esterl, “Completion of the    primary structure of human high-molecular-mass kininogen. The amino    acid sequence of the entire heavy chain and evidence for its    evolution by gene triplication,” Eur. J. Biochem., 154(2):471–478,    1986.-   Kenyon, Browne, D'Amato, “Effects of thalidomide and related    metabolites in a mouse corneal model of neovascularization,” Exp.    Eye Res., 64(6):971–978, 1997.-   Kim, Kwak, Ahn, So, Liu, Koh, Koh, “Molecular cloning and    characterization of a novel angiopoietin family protein,    angiopoietin-3”, FEBS Lett., 443(3):353–6, 1999.-   Kitamura, Takagaki, Furuto, Tanaka, Nawa, Nakanishi, “A single gene    for bovine high molecular weight and low molecular weight    kininogens,” Nature, 305(5934):545–549, 1983.-   Kitamura, Kitagawa, Fukushima, Takagaki, Miyata, Nakanishi,    “Structural organization of the human kininogen gene and a model for    its evolution,” J. Biol. Chem., 260(14):8610–8617, 1985.-   Kitamura, Ohkubo, Nakanishi, “Molecular biology of the    angiotensinogen and kininogen genes,” J. Cardiovasc. Pharmacol.,    10(Suppl 7):S49–S53, 1987.-   Kitamura, Nawa, Takagaki, Furuto-Kato, Nakanishi, “Cloning of cDNAs    and genomic DNAs for high-molecular-weight and low-molecular-weight    kininogens,” Methods Enzymol., 163:230–240, 1988.-   Kleinman et al., “The laminins: a family of basement membrane    glycoproteins important in cell differentiation and tumor    metastases,” Vitam. Horm., 47:161–186, 1993.-   Kohler and Milstein, “Continuous cultures of fused cells secreting    antibody of predefined specificity,” Nature, 256:495–497, 1975.-   Kohler and Milstein, “Derivation of specific antibody-producing    tissue culture and tumor lines by cell fusion,” Eur. J. Immunol.,    6:511–519, 1976.-   Konieczny, Bobrzecka, Laidler, Rybarska, “The combination of IgM    subunits and proteolytic IgG fragment by controlled formation of    interchain disulphides,” Haematologia, 14(1):95–99, 1981.-   Kuzu, Bicknell, Fletcher, Gatter, “Expression of adhesion molecules    on the endothelium of normal tissue vessels and vascular tumors,”    Lab. Invest., 69(3):322–328, 1993.-   Kyte and Doolittle, “A simple method for displaying the hydropathic    character of a protein,” J. Mol. Biol., 157(1):105–132, 1982.-   Lane, Iruela-Arispe, Sage, “Regulation of gene expression by SPARC    during angiogenesis in vitro. Changes in fibronectin,    thrombospondin-1, and plasminogen activator inhibitor-1,” J. Biol.    Chem., 267(23):16736–16745, 1992.-   Lee et al., “Inhibition of urokinase activity by the antiangiogenic    factor 16K prolactin: activation of plasminogen activator inhibitor    1 expression,” Endocrinology, 139(9):3696–3703, 1998.-   Leppink, Bishop, Sedmak, Henry, Ferguson, Streeter, Butcher, Orosz,    “Inducible expression of an endothelial cell antigen on murine    myocardial vasculature in association with interstitial cellular    infiltration,” Transplantation, 48(5):874–877, 1989.-   Levy, Gharavi, Sammaritano, Habina, Lockshin, “Fatty acid chain is a    critical epitope for antiphospholipid antibody,” J. Clin. Immunol.,    10(3): 141–145, 1990.-   Lin, Shroyer, Walter, Lyden, Ng, Rote, “Monoclonal IgM    antiphosphatidylserine antibody reacts against cytoskeleton-like    structures in cultured human umbilical cord endothelial cells,”    Am. J. Reprod. Immun., 33:97–107, 1995.-   Lin, Buxton, Acheson, Radziejewski, Maisonpierre, Yancopoulos,    Channon, Hale, Dewhirst, George, Peters, “Anti-angiogenic gene    therapy targeting the endothelium-specific receptor tyrosine kinase    Tie2”, Proc. Natl. Acad. Sci., USA, 95(15):8829–34, 1998.-   Lindner and Borden, “Effects of tamoxifen and interferon-beta or the    combination on tumor-induced angiogenesis,” Int. J. Cancer,    71(3):456–461, 1997.-   Lingen, Polyerini, Bouck, “Inhibition of squamous cell carcinoma    angiogenesis by direct interaction of retinoic acid with endothelial    cells,” Lab. Invest., 74(2):476–483, 1996.-   Lingen, Polyerini, Bouck, “Retinoic acid and interferon alpha act    synergistically as antiangiogenic and antitumor agents against human    head and neck squamous cell carcinoma,” Cancer Res.,    58(23):5551–5558, 1998.-   Liu, Moy, Kim, Xia, Rajasekaran, Navarro, Knudsen, Bander,    “Monoclonal antibodies to the extracellular domain of    prostate-specific membrane antigen also react with tumor vascular    endothelium”, Cancer Res., 57:3629–3634, 1997.-   Majewski et al., “Vitamin D3 is a potent inhibitor of tumor    cell-induced angiogenesis,” J. Investig. Dermatol. Symp. Proc.,    1(1):97–101, 1996.-   Mandriota and Pepper, “Regulation of angiopoietin-2 mRNA levels in    bovine microvascular endothelial cells by cytokines and hypoxia”,    Circ. Res., 83(8):852–9, 1998.-   Maneta-Peyret, Bessoule, Geffard, Cassagne, “Demonstration of high    specificity antibodies against phosphatidylserine,” J. Immun. Meth.,    108:123–127, 1988.-   Maneta-Peyret, Freyburger, Bessoule, Cassagne, “Specific    immunocytochemical visualization of phosphatidylserine,” J. Immun.    Methods, 122:155–159, 1989.-   Manetti et al., “Synthesis and binding mode of heterocyclic    analogues of suramin inhibiting the human basic fibroblast growth    factor,” Bioorg. Med. Chem., 6(7):947–958, 1998.-   Martin, Reutelingsperger, McGahon, Rader, van Schie, LaFace, Green,    “Early redistribution of plasma membrane phosphatidylserine is a    general feature of apoptosis regardless of the initiating stimulus:    inhibition by overexpression of Bc-1–2 and Abl,” J. Exp. Med.,    182(5):1545–1556, 1995.-   Matsuura, Igarashi, Yasuda, Triplett, Koike, “Anticardiolipin    antibodies recognize beta 2-glycoprotein I structure altered by    interacting with an oxygen modified solid phase surface,” J. Exp.    Med, 179(2):457–462, 1994.-   McNeil, Simpson, Chesterman, Krilis, “Anti-phospholipid antibodies    are directed against a complex antigen that includes a lipid-binding    inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H),”    Proc. Natl. Acad. Sci. USA, 87(11):4120–4124, 1990.-   Mills, Brooker, Camerini-Otero, “Sequences of human immunoglobulin    switch regions: implications for recombination and transcription,”    Nucl. Acids Res., 18:7305–7316, 1990.-   Miyake, Medina, Ishihara, Kimoto, Auerbach, Kincade, “VCAM-like    adhesion molecule on murine bone marrow stromal cells mediates    binding of lymphocyte precursors in culture,” J. Cell. Biol.,    114:557–565, 1991.-   Moldovan, Moldovan, Simionescu, “Binding of vascular anticoagulant    alpha (annexin V) to the aortic intima of the hypercholesterolemic    rabbit. An autoradiographic study,” Blood Coagul Fibrinolysis,    5(6):921–928, 1994.-   Montesano, Pepper, Mohle-Steinlein, Risau, Wagner, Orci, “Increased    proteolytic activity is responsible for the aberrant morphogenetic    behavior of endothelial cells expressing the middle T oncogene,”    Cell, 62:435–445, 1990.-   Moore et al., “Tumor angiogenesis is regulated by CXC    chemokines,” J. Lab. Clin. Med., 132(2):97–103, 1998.-   Morrison, Johnson, Herzenberg, Oi, “Chimeric human antibody    molecules: mouse antigen-binding domains with human constant region    domains,” Proc. Natl. Acad. Sci. USA, 81(21):6851–6855, 1984.-   Morrison, Wims, Kobrin, Oi, “Production of novel immunoglobulin    molecules by gene transfection,” Mt. Sinai J. Med., 53(3):175, 1986.-   Morrissey, Fair, Edgington, “Monoclonal antibody analysis of    purified and cell-associated tissue factor,” Thromb. Res.,    52:247–261, 1988.-   Müller, Pomorski, Müller, Zachowski, Herrmann, “Protein-dependent    translocation of aminophospholipids and asymmetric transbilayer    distribution of phospholipids in the plasma membrane of ram sperm    cells,” Biochemistry, 33:9968–9974, 1994.-   Munro, “Endothelial-leukocyte adhesive interactions in inflammatory    diseases,” European. Heart Journal, 14:72–77, 1993.-   Murphy, Joseph, Stephens, Horrocks, “Phospholipid composition of    cultured human endothelial cells,” Lipids, 27(s):150–153, 1992.-   Murray, Clauss, Thurston, Stern, “Tumour-derived factors which    induce endothelial tissue factor and enhance the procoagulant    response to TNF,” Int. J. Radiat. Biol., 60(1–2):273–277, 1991.-   Nagler, Feferman, Shoshan, “Reduction in basic fibroblast growth    factor mediated angiogenesis in vivo by linomide,” Connect Tissue    Res., 37(1–2):61–68, 1998.-   Nakamura, Shidara, Kawaguchi, Azuma, Mitsuda, Onishi, Yamaji, Wada,    “Lupus anticoagulant autoantibody induces apoptosis in umbilical    vein endothelail cells: involvement of annexin V,” Biocehm. Biophys.    Res. Comm., 205(2):1488–1493, 1994.-   Nakamura, Ban, Yamaji, Yoneda, Wada, “Localization of the    apoptosis-inducing activity of lupus anticoagulant in an annexin    V-binding antibody subset,” J. Clin. Invest., 101(9):1951–1959,    1998.-   Nakanishi, Ohkubo, Nawa, Kitamura, Kageyama, Ujihara,    “Angiotensinogen and kininogen: closing and sequence analysis of the    cDNAs,” Clin. Exp. Hypertens., 5(7–8):997–1003, 1983.-   Nawroth and Stern, “Modulation of endothelial cell hemostatic    properties by tumor necrosis factor,” J. Exp. Med., 163:740–745,    1986.-   Nawroth, Stern, Kisiel, Bach, “Cellular requirements for tissue    factor generation by bovine aortic endothelial cells in culture,”    Thromb. Res., 40:677–691, 1985.-   Nawroth, Handley, Matsueda, DeWaal, Gerlach, Blohm, Stern, “Tumor    necrosis factor/cachectin-induced intravascular fibrin formation in    meth A fibrosarcomas,” J. Exp. Med., 168:637–647, 1988.-   Obringer, Rote, Walter, “Antiphospholipid antibody binding to    bilayer-coated glass microspheres,” J. Immun. Methods, 185:81–93,    1995.-   Ogawa, Shreeniwas, Brett, Clauss, Furie, Stern, “The effect of    hypoxia on capillary endothelial cell function: modulation of    barrier and coagulant function,” J. Haematology, 75:517–524, 1990.-   Ohizumi, Tsunoda, Taniguchi, Saito, Esaki, Makimoto, Wakai,    Tsutsumi, Nakagawa, Utoguchi, Kaiho, Ohsugi, Mayumi, “Antibody-based    therapy targeting tumor vascular endothelial cells suppresses solid    tumor growth in rats,” Biochem. Biophys. Res. Comm., 236:493–496,    1997.-   Oikawa et al., “A highly potent antiangiogenic activity of    retinoids,” Cancer Lett., 48(2): 157–162, 1989.-   O'Reilly et al., “Angiostatin: a novel angiogenesis inhibitor that    mediates the suppression of metastases by a Lewis lung carcinoma,”    Cell, 79:315–328, 1994.-   O'Reilly et al., “Endostatin: an endogenous inhibitor of    angiogenesis and tumor growth,” Cell, 88(2):277–285, 1997.-   Ortel, Devore-Carter, Quinn-Allen, Kane, “Deletion analysis of    recombinant human factor V. Evidence for a phosphatidylserine    binding site in the second C-type domain,” J. Biol. Chem.,    267:4189–4198, 1992.-   Papapetropoulos, Garcia-Cardena, Dengler, Maisonpierre, Yancopoulos,    Sessa, “Direct actions of angiopoietin-1 on human endothelium:    evidence for network stabilization, cell survival, and interaction    with other angiogenic growth factors”, Lab. Invest., 79(2):213–23,    1999.-   Parmley and Smith, “Antibody-selectable filamentous fd phage    vectors: affinity purification of target genes,” Gene,    73(2):305–318, 1988.-   Patey, Vazeux, Canioni, Potter, Gallatin, Brousse, “Intercellular    adhesion molecule-3 on endothelial cells: Expression in tumors but    not in inflammatory responses,” Am. J. Pathol., 148:465–472, 1996.-   Pepper et al., “Leukemia inhibitory factor (LIF) inhibits    angiogenesis in vitro,” J. Cell Sci., 108(Pt 1):73–83, 1995.-   Perry, Hall, Bell, Jones, “Sequence analysis of a mammalian    phospholipid-binding protein from testis and epididymis and its    distribution between spermatozoa and extracellular secretions,”    Biochem. J, 301(Pt 1):235–242, 1994.-   Qamar, Gharavi, Levy, Lockshin, “Lysophosphatidylethanolamine is the    antigen to which apparent antibody to phosphatidylethanolamine    binds,” J. Clin. Immunol., 10(4):200–203, 1990.-   Qu, Conroy, Walker, Wooding, Lucy, “Phosphatidylserine-mediated    adhesion of T-cells to endothelial cells,” J. Biochem., 317(Pt    2):343–346, 1996.-   Quinn et al., CM101, a polysaccharide antitumor agent, does not    inhibit wound healing in murine models,” J. Cancer Res. Clin.    Oncol., 121(4):253–256, 1995.-   Rao, Tait, Hoang, “Binding of annexin V to a human ovarian carcinoma    cell line (OC-2008). Contrasting effects on cell surface factor    VIIa/tissue factor activity and prothrombinase activity,” Thromb.    Res., 67(5):517–531, 1992.-   Rauch and Janoff, “Phospholipid in the hexagonal II phase is    immunogenic: evidence for immunorecognition of nonbilayer lipid    phases in vivo,” Proc. Natl. Acad. Sci., USA, 87(11):4112–4114,    1990.-   Rauch, Tannenbaum, Tannenbaum, Ramelson, Cullis, Tilcock, Hope,    Janoff, “Human hybridoma lupus anticoagulants distinguish between    lamellar and hexagonal phase lipid systems,” J. Biol. Chem.,    261(21):9672–9677, 1986.-   Ravanat, Archipoff, Beretz, Freund, Cazenave, Freyssinet, “Use of    annexin-V to demonstrate the role of phosphatidylserine exposure in    the maintenance of hemostatic balance by endothelial cells,”    Biochem. J., 282:7–13, 1992.-   RayChaudhury and D'Amore, “Endothelial cell regulation by    transforming growth factor-beta,” J. Cell Biochem., 47(3):224–229,    1991.-   Richer and Lo, “Introduction of human DNA into mouse eggs by    injection of dissected human chromosome fragments”, Science 245,    175–177, 1989.-   Riechmann, Clark, Waldmann, Winter, “Reshaping human antibodies for    therapy,” Nature, 332(6162):323–327, 1988.-   Rote, “Antiphospholipid antibodies and recurrent pregnancy loss,”    Am. J. Reprod. Immun., 35:394–401, 1996.-   Rote, Ng, Dostal-Johnson, Nicholson, Siekman, “Immunologic detection    of phosphatidylserine externalization during thrombin-induced    platelet activation,” Clin. Immunol. Immunopathol., 66:193–200,    1993.-   Rote, Chang, Katsuragawa, Ng, Lyden, Mori, “Expression of    phosphatidylserine-dependent antigens on the surface of    differentiating BeWo human choriocarcinoma cells,” Am. J. Reprod.    Immun., 33:114–121, 1995.-   Ruf and Edgington, “Structural biology of tissue factor, the    initiator of thrombogenesis in vivo,” FASEB J., 8:385–390, 1994.-   Ruf, Rehemtulla, Edgington, “Phospholipid-independent and -dependent    interactions required for tissue factor receptor and cofactor    function,” Biol. Chem., 266:2158–2166, 1991.-   Sakamoto et al, “Heparin plus cortisone acetate inhibit tumor growth    by blocking endothelial cell proliferation,” Canc. J, 1:55–58, 1986.-   Sambrook, Fritsch, Maniatis, Molecular Cloning. A Laboratory Manual,    2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.-   Sang, “Complex role of matrix metalloproteinases in angiogenesis,”    Cell Res., 8(3):171–177, 1998.-   Schick, “The organization of aminophospholipids in human platelet    membranes: selective changes induced by thrombin”, J. Lab. Clin.    Med., 91(5):802–810, 1978.-   Schick, Kurica, Chacko, “Location of phosphatidylethanolamine and    phosphatidylserine in the human platelet plasma membrane,” J. Clin.    Invest., 57:1221–1226, 1976.-   Schoentgen, Saccoccio, Jolles, Bemier, Jolles, “Complete amino acid    sequence of a basic 21-kDa protein from bovine brain cytosol,”    Eur. J. Biochem., 166(2):333–338, 1987.-   Schorer, Rick, Swaim, Moldow, “Structural features of endotoxin    required for stimulation of endothelial cell tissue factor    production; exposure of preformed tissue factor after    oxidant-mediated endothelial cell injury,” J. Lab. Clin. Med.,    106:38–42, 1985.-   Schuurmans Stekhoven, Tijmes, Umeda, Inoue, De Pont, “Monoclonal    antibody to phosphatidylserine inhibits Na⁺/K⁺-ATPase activity,”    Biochimica et Biophysica Acta, 1194:155–165, 1994.-   Sheibani and Frazier, “Thrombospondin 1 expression in transformed    endothelial cells restores a normal phenotype and suppresses their    tumorigenesis,” Proc. Natl. Acad. Sci. USA, 92(15):6788–6792, 1995.-   Sheu et al., “Inhibition of angiogenesis in vitro and in vivo:    comparison of the relative activities of triflavin, an    Arg-Gly-Asp-containing peptide and anti-alpha(v)beta3 integrin    monoclonal antibody,” Biochim. Biophys. Acta, 1336(3):445–454, 1997.-   Shyu, Manor, Magner, Yancopoulos, Isner, “Direct intramuscular    injection of plasmid DNA encoding angiopoietin-1 but not    angiopoietin-2 augments revascularization in the rabbit ischemic    hindlimb”, Circulation, 98(19):2081–7, 1998.-   Sideras, Mizuta, Kanamori, Suzuki, Okamoto, Kuze, Ohno, Doi,    Fukuhara, Hassan, et al., “Production of sterile transcripts of C    gamma genes in an IgM-producing human neoplastic B cell line that    switches to IgG-producing cells,” Intl. Immunol., 1(6):631–642,    1989.-   Sipos et al., “Inhibition of tumor angiogenesis,” Ann. NY Acad.    Sci., 732:263–272, 1994.-   Sluiter, Pietersma, Lamers, Koster, “Leukocyte adhesion molecules on    the vascular endothelium: their role in the pathogenesis of    cardiovascular disease and the mechanisms underlying their    expression,” J. Cardiol. Pharmacol., 22:S37–S44, 1993.-   Smirnov, Triplett, Comp, Esmon, Esmon, “On the role of    phosphatidylethanolamine in the inhibition of activated protein C    activity by antiphospholipid antibodies,” J. Clin. Invest.,    95(1):309–316, 1995.-   Soff et al., “Expression of plasminogen activator inhibitor type I    by human prostate carcinoma cells inhibits primary tumor growth,    tumor-associated angiogenesis, and metastasis to lung and liver in    an athymic mouse model,” J. Clin. Invest., 96(6):2593–2600, 1995.-   Staal-van den Brekel, Thunnissen, Buurman, Wouters, “Expression of    E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular    cell adhesion molecule (VCAM)-1 in non-small-cell lung carcinoma,”    Virchows Arch., 428:21–27, 1996.-   Staub, Harris, Khamashta, Savidge, Chahade, Hughes, “Antibody to    phosphatidylethanolamine in a patient with lupus anticoagulant and    thrombosis,” Ann. Rheum. Dis., 48(2):166–169, 1989.-   Stone, Ruf, Miles, Edgington, Wright, “Recombinant soluble human    tissue factor secreted by Saccharomyces cerevisiae and refolded    from E. coli inclusion bodies: glycosylation of mutants, activity,    and physical characterization,” Biochem. J., 310(2):605–614, 1995.-   Stout, Basse, Luhm, Weiss, Wiedmer, Sims, “Scott syndrome    erythrocytes contain a membrane protein capable of mediating    Ca²⁺-dependent transbilayer migration of membrane phospholipids,” J.    Clin. Invest., 99(9):2232–2238, 1997.-   Stratmann, Risau, Plate, “Cell type-specific expression of    angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma    angiogenesis”, Am. J. Pathol., 153(5): 1333–9, 1998.-   Sugi and McIntyre, “Autoantibodies to phosphatidylethanolamine (PE)    recognize a kininogen-PE complex,” Blood, 86(8):3083–3089, 1995.-   Sugi and McIntyre, “Phosphatidylethanolamine induces specific    conformational changes in the kininogens recognizable by    antiphosphatidylethanolamine antibodies,” Thromb. Haemost.,    76(3):354–360, 1996a.-   Sugi and McIntyre, “Autoantibodies to kininogen-    phosphatidylethanolamine complexes augment thrombin-induced platelet    aggregation,” Thromb. Res., 84(2):97–109, 1996b.-   Sugimura, Donato, Kakar, Scully, “Annexin V as a probe of the    contribution of anionic phospholipids to the procoagulant activity    of tumor cell surfaces,” Blood Coagul. Fibrinolysis, 5(3):365–373,    1994.-   Tada et al., “Inhibition of tubular morphogenesis in human    microvascular endothelial cells by co-culture with chondrocytes and    involvement of transforming growth factor beta: a model for    avascularity in human cartilage,” Biochim. Biophys. Acta,    1201(2):135–142, 1994.-   Takano et al., “Suramin, an anticancer and angiosuppressive agent,    inhibits endothelial cell binding of basic fibroblast growth factor,    migration, proliferation, and induction of urokinase-type    plasminogen activator,” Cancer Res., 54(10):2654–2660, 1994.-   Tanaka et al., “Viral vector-mediated transduction of a modified    platelet factor 4 cDNA inhibits angiogenesis and tumor growth,” Nat.    Med., 3(4):437–442, 1997.-   Tanaka, Mori, Sakamoto, Makuuchi, Sugimachi, Wands, “Biologic    significance of angiopoietin-2 expression in human hepatocellular    carcinoma”, J. Clin. Invest., 103(3):341–5, 1999.-   Thornhill, Kyan-Aung, Haskard, “IL-4 increases human endothelial    cell adhesiveness for T cells but not for neutrophils,” J. Immunol.,    144:3060–3065, 1990.-   Thorpe et al., “Heparin-Steroid Conjugates: New Angiogenesis    Inhibitors with Antitumor Activity in Mice,” Cancer Res.,    53:3000–3007, 1993.-   Tolsma et al., “Peptides derived from two separate domains of the    matrix protein thrombospondin-1 have anti-angiogenic activity,” J.    Cell Biol., 122(2):497–511, 1993.-   Toti, Satta, Fressinaud, Meyer, Freyssinet, “Scott syndrome,    characterized by impaired transmembrane migration of procoagulant    phosphatidylserine and hemorrhagic complications, is an inherited    disorder,” Blood, 87(4):1409–1415, 1996.-   Trudell, Ardies, Anderson, “Antibodies raised against    trifluoroacetyl-protein adducts bind to    N-trifluoroacetyl-phosphatidylethanolamine in hexagonal phase    phospholipid micelies,” J. Pharmacol. Exp. Ther., 257(2):657–662,    1991a.-   Trudell, Ardies, Green, Allen, “Binding of anti-acetaldehyde IgG    antibodies to hepatocytes with an    acetaldehyde-phosphatidylethanolamine adduct on their surface,”    Alcohol Clin. Exp. Res., 15(2):295–299, 1991b.-   Tryggvason, “The laminin family,” Curr. Opin. Cell Biol.,    5(5):877–882, 1993.-   Umeda, Igarashi, Nam, Inoue, “Effective production of monoclonal    antibodies against phosphatidylserine: Stereo-specific recognition    of phosphatidylserine by monoclonal antibody,” J. Immun.,    143(7):2273–2279, 1989.-   Utsugi, Schroit, Connor, Bucana, Fidler, “Elevated expression of    phosphatidylserine in the outer membrane leaflet of human tumor    cells and recognition by activated human blood monocytes,” Cancer    Res., 51(11):3062–3066, 1991.-   Valenzuela, Griffiths, Rojas, Aldrich, Jones, Zhou, McClain,    Copeland, Gilbert, Jenkins, Huang, Papadopoulos, Maisonpierre,    Davis, Yancopoulos, “Angiopoietins 3 and 4: diverging gene    counterparts in mice and humans”, Proc. Natl. Acad. Sci., USA,    96(5):1904–9, 1999.-   van Dijk, Warnaar, van Eendenburg, Thienpont, Braakman, Boot,    Fleuren, Bolhuis, “Induction of tumor-cell lysis by bi-specific    monoclonal antibodies recognizing renal-cell carcinoma and CD3    antigen,” Int. J. Cancer, 43:344–349, 1989.-   Van Heerde, Poort, van T Veer, Reutelingsperger, de Groot, “Binding    of recombinant annexin V to endothelial cells: effect of annexin V    binding on endothelial-cell-mediated thrombin formation,” J.    Biochem., 302:305–312, 1994.-   Vermes, Haanes, Steffens-Nakken, Reutelingsperger, “A novel assay    for apoptosis. Flow cytometric detection of phosphatidylserine    expression on early apoptotic cells using fluorescein labeled    Annexin V,” J. Immunol. Methods, 184(1):39–51, 1995.-   Vitetta et al., “Phase I immunotoxin trial in patients with B-cell    lymphoma,” Cancer Res., 15:4052–4058, 1991.-   Vlachoyiannopoulos, Beigbeder, Duelanes, Youinou, Hunt, Krilis,    Moutsopoulos, “Antibodies to phosphatidylethanolamine in    antiphospholipid syndrome and systemic lupus erythematosus: their    correlation with anticardiolipin antibodies and beta 2    glycoprotein-I plasma levels,” Autoimmunity, 16(4):245–249, 1993.-   Vogt, Ng, Rote, “A model for the antiphospholipid antibody syndrome:    Monoclonal antiphosphatidylserine antibody induces intrauterine    growth restriction in mice,” Am. J. Obstet. Gynecol., 174:700–707,    1996.-   Vogt, Ng, Rote, “Antiphosphatidylserine antibody removes Annexin V    and facilitates the binding prothrombin at the surface of a    choriocarcinoma model of trophoblast differentiation,” Am. J.    Obstet. Gynecol., 177:964–972, 1997.-   Volpert, Lawler, Bouck, “A human fibrosarcoma inhibits systemic    angiogenesis and the growth of experimental metastases via    thrombospondin-1,” Proc. Natl. Acad. Sci. USA, 95(11):6343–6348,    1998.-   Vukanovic et al., “Antiangiogenic effects of the    quinoline-3-carboxamide linomide,” Cancer Res., 53(8):1833–1837,    1993.-   Waltenberger et al., “Suramin is a potent inhibitor of vascular    endothelial growth factor. A contribution to the molecular basis of    its antiangiogenic action,” J. Mol. Cell Cardiol., 28(7):1523–1529,    1996.-   Wamil et al., “Soluble E-selectin in cancer patients as a marker of    the therapeutic efficacy of CM101, a tumor-inhibiting    anti-neovascularization agent, evaluated in phase I clinical    trail,” J. Cancer Res. Clin. Oncol., 123(3):173–179, 1997.-   Wells, “Starving cancer into submission”, Chem. Biol., 5(4):R87–88,    1998.-   Weiss, Young, LoBuglio, Slivka and Nimeh, “Role of Hydrogen Peroxide    in Neutrophil-Mediated Destruction of Cultured Endothelial    Cells,” J. Clin. Invest., 68:714–721, 1981.-   White, Handler, Smith, Hill, Lehman, In: Principles of Biochemistry,    6th Edition, McGraw-Hill, Inc. N.Y., Chapter 3, pp. 48–54, 1978.-   Williamson and Schlegel, “Back and forth: the regulation and    function of transbilayer phospholipid movement in eukaryotic cells,”    Molec. Mem. Biol., 11: 199–216, 1994.-   Winter and Milstein, “Man-made antibodies,” Nature, 349:293–299,    1991.-   Wolff et al., “Dexamethasone inhibits glioma-induced formation of    capillary like structures in vitro and angiogenesis in vivo,” Klin.    Padiatr., 209(4):275–277, 1997.-   Yamada, Moldow, Sacks, Craddock, Boogaens and Jacob, “Deleterious    Effects of Endotoxin on Cultured Endothelial Cells: An in vitro    Model of Vascular injury,” Inflammation, 5:115–116, 1981.-   Yamamura et al, “Effect of Matrigel and laminin peptide YIGSR on    tumor growth and metastasis,” Semin. Cancer Biol., 4(4):259–265,    1993.-   Yoon et al., “Inhibitory effect of Korean mistletoe (Viscum album    coloratum) extract on tumour angiogenesis and metastasis of    haematogenous and non-haematogenous tumour cells in mice,” Cancer    Lett, 97(1):83–91, 1995.-   Yoshida et al., “Suppression of hepatoma growth and angiogenesis by    a fumagillin derivative TNP470: possible involvement of nitric oxide    synthase,” Cancer Res., 58(16):3751–3756, 1998.-   Zacharski, Memoli, Ornstein, Rousseau, Kisiel, Kudryk, “Tumor cell    procoagulant and urokinase expression in carcinoma of the ovary,” J.    Natl. Cancer Inst., 85:1225–1230, 1993.-   Zhao, Zhou, Wiedmer, Sims, “Level of expression of phospholipid    scramblase regulates induced movement of phosphatidylserine to the    cell surface,” J. Biol. Chem., 273:6603–6606, 1998.-   Zhou, Zhao, Stout, Luhm, Wiedmer, Sims, “Molecular cloning of human    plasma membrane phospholipid scramblase. A protein mediating    transbilayer movement of plasma membrane phospholipids,” J. Biol.    Chem., 272(29):18240–18244, 1997.-   Ziche et al., “Linomide blocks angiogenesis by breast carcinoma    vascular endothelial growth factor transfectants,” Br. J. Cancer,    77(7):1123–1129, 1998.-   Zwaal, Bevers, Comfurius, Rosing, Tilly, Verhallen, “Loss of    membrane phospholipid asymmetry during activation of blood platelets    and sickled red cells; mechanisms and physiological significance,”    Mol. Cell. Biochem., 91:23–31, 1989.-   Zwaal, Comfurius, Bevers, “Platelet procoagulant activity and    microvesicle formation. Its putative role in hemostasis and    thrombosis,” Biochimica et Biophysica Acta, 1180:1–8, 1992.

1. A kit comprising, in a pharmaceutically acceptable form,therapeutically effective amounts of: (a) at least a first anticanceragent, wherein said at least a first anti-cancer agent is at least afirst targeting agent-therapeutic agent construct that comprises atleast a first targeting agent operatively attached to at least a firsttherapeutic agent, wherein said at least a first targeting agent bindsto phosphatidylethanolamine expressed on the luminal surface of bloodvessels of a vascularized tumor, and (b) at least a second anti canceragent other than said at least a first targeting agent-therapeutic agentconstruct.
 2. A kit comprising, in a pharmaceutically acceptable form,therapeutically effective amounts of: (a) at least a first anti-canceragent, wherein said at least a first anticancer agent is at least afirst targeting agent-therapeutic agent construct that comprises atleast a first targeting agent operatively attached to at least a firsttherapeutic agent, wherein said at least a first targeting agent bindsto phosphatidylethanolamine on the luminal surface of blood vessels of avascularized tumor; and (b) at least a second anti-cancer agent otherthan said at least a first targeting agent-therapeutic agent construct;wherein said at least a second anti-cancer agent: (i) increasesphosphatidylethanolamine expression in the endothelium of said bloodvessels of said vascularized tumor or injures or induces apoptosis inthe endothelium of said blood vessels of said vascularized tumor, or(ii) kills tumor cells of said tumor or is an anti-angiogenic agent thatinhibits metastasis of tumor cells.
 3. The kit of claim 2, wherein saidat least a second anti-cancer agent increases phosphatidylethanolamineexpression in the endothelium of said blood vessels of said vascularizedtumor or injures or induces apoptosis in the endothelium of said bloodvessels of said vascularized tumor.
 4. The kit of claim 3, wherein saidat least a second anti-cancer agent is taxol, vincristine, vinblastine,neomycin, a combretastatin, a podophyllotoxin, TNF-α, angiostatin,endostatin, vasculostatin, an α_(v)β₃ antagonist, a calcium-fluxinducing agent, a calcium ionophore, H₂O₂, thrombin, an inflammatorycytokine or interleukin-4.
 5. The kit of claim 2, wherein said at leasta second anti-cancer agent kills tumor cells of said tumor or is ananti-angiogenic agent that inhibits metastasis of tumor cells.
 6. Thekit of claim 5, wherein said at least a second anti-cancer agent is ananti-tumor cell immunoconjugate, a chemotherapeutic agent or ananti-angiogenic agent.
 7. The kit of claim 1, wherein said targetingagent-therapeutic agent construct comprises at least a firstanti-aminophospholipid antibody that binds to phosphatidylethanolamine,or antigen-binding fragment thereof.
 8. The kit of claim 7, wherein saidtargeting agent-therapeutic agent construct comprises at least a firstIgG or IgM anti-aminophospholipid antibody that binds tophosphatidylethanolamine.
 9. The kit of claim 7, wherein said targetingagent-therapeutic agent construct comprises at least a first scFv, Fv,Fab′, Fab or F(ab′)₂ antigen-binding fragment of ananti-aminophospholipid antibody that binds to phosphatidylethanolamine.10. The kit of claim 7, wherein said targeting agent-therapeutic agentconstruct comprises at least a first recombinant anti-aminophospholipidantibody that binds to phosphatidylethanolamine, or antigen-bindingfragment thereof.
 11. The kit of claim 7, wherein said targetingagent-therapeutic agent construct comprises at least a first human,humanized or part-human chimeric anti-aminophospholipid antibody thatbinds to phosphatidylethanolamine, or antigen-binding fragment thereof.12. The kit of claim 7, wherein said targeting agent-therapeutic agentconstruct comprises at least a first monoclonal anti-aminophospholipidantibody that binds to phosphatidylethanolamine, or antigen-bindingfragment thereof.
 13. The kit of claim 1, wherein said targetingagent-therapeutic agent construct comprises at least a firstaminophospholipid binding protein that binds to phosphatidylethanolamineor an aminophospholipid-binding fragment thereof.
 14. The kit of claim13, wherein said targeting agent-therapeutic agent construct comprisesat least a first kininogen or a phosphatidylethanolamine-bindingfragment thereof.
 15. The kit of claim 1, wherein said targetingagent-therapeutic agent construct comprises at least a firstanticellular or cytotoxic agent.
 16. The kit of claim 15, wherein saidtargeting agent-therapeutic agent construct comprises at least a firstgelonin, ricin A chain or deglycosylated ricin A chain.
 17. The kit ofclaim 1, wherein said targeting agent-therapeutic agent constructcomprises at least a first coagulant.
 18. The kit of claim 17, whereinsaid targeting agent-therapeutic agent construct comprises at least afirst Tissue Factor, dimeric Tissue Factor, trimeric Tissue Factor,polymeric Tissue Factor, mutant Tissue Factor, truncated Tissue Factoror a Tissue Factor derivative.
 19. The kit of claim 1, wherein saidtargeting agent-therapeutic agent construct comprises an antibody thatbinds to phosphatidylethanolamine, or antigen binding fragment thereofwherein said antibody or antigen binding fragment thereof is directly orindirectly attached to truncated Tissue Factor.
 20. The kit of claim 1,wherein said kit comprises at least a first pharmaceutically acceptableformulation suitable for intravenous administration.
 21. The kit ofclaim 1, wherein said at least a first targeting agent-therapeutic agentconstruct and said at least a second anticancer agent are comprisedwithin a single pharmaceutical composition.
 22. The kit of claim 1,wherein said at least a first targeting agent-therapeutic agentconstruct and said at least a second anti-cancer agent are comprisedwithin distinct pharmaceutical compositions.
 23. The kit of claim 1,wherein said at least a second anti-cancer agent is a chemotherapeuticagent, radiotherapeutic agent, anti-angiogenic agent orapoptosis-inducing agent.
 24. The kit of claim 1, wherein said at leasta second anti-cancer agent is an antibody-therapeutic agent constructcomprising a second targeting antibody, or antigen-binding fragmentthereof, that binds to a surface-expressed, surface-accessible orsurface-localized component of a tumor cell, tumor stroma or tumorvasculature; wherein said targeting antibody or fragment thereof isoperatively linked to a therapeutic agent.
 25. The kit of claim 24,wherein said second targeting antibody, or antigen-binding fragmentthereof, binds to a surface-expressed, surface-accessible,surface-localized, cytokine-inducible or coagulant-inducible componentof intratumoral blood vessels of a vascularized tumor.
 26. The kit ofclaim 25, wherein said second targeting antibody, or anti-bindingfragment thereof, binds to a component of intratumoral vasculatureselected from the group consisting of an aminophospholipid, endoglin, aTGFβ receptor, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a VEGF/VPFreceptor, an FGF receptor, a TIE, α_(v)β₃ integrin, pleiotropin,endosialin, an MHC Class II protein, VEGF/VPF, FGF, TGFβ, a ligand thatbinds to a TIE, a tumor-associated fibronectin isoform, scatterfactor/hepatocyte growth factor (HGF), platelet factor 4 (PF4), PDGF andTIMP.
 27. The kit of claim 24, wherein said second targeting antibody,or antigen-binding fragment thereof, is operatively linked to gelonin,deglycosylated ricin A chain, Tissue Factor, truncated Tissue Factor orto an antibody, or antigen-binding fragment thereof, that binds toTissue Factor or truncated Tissue Factor.
 28. The kit of claim 1,wherein said kit further comprises a targeting agent-detectable agentconstruct that comprises a second targeting agent that binds tophosphatidylethanolamine operatively attached to a detectable agent. 29.In combination, biologically effective amounts of: (a) a firstcomposition comprising at least a first anti cancer agent, wherein saidat least a first anti-cancer agent is at least a first targetingagent-therapeutic agent construct that comprises at least a firsttherapeutic agent operatively attached to at least a first targetingagent that binds to phosphatidylethanolamine; (b) a second compositioncomprising a targeting agent-detectable agent construct that comprises adetectable agent operatively attached to a second targeting agent thatbinds to phosphatidylethanolamine; and (c) at least a second anti-canceragent other than said at least a first targeting agent-therapeutic agentconstruct.
 30. The kit of claim 29, wherein the targeting agent of saidat least a first targeting agent-therapeutic agent construct and thetargeting agent of said targeting agent-detectable agent construct areanti-aminophospholipid antibodies that bind to phosphatidylethanolamine,or antigen-binding fragments thereof, obtained from the same antibodypreparation or antibody-producing hybridoma.
 31. The combination ofclaim 29, wherein said first composition is a pharmaceuticalcomposition.
 32. The combination of claim 29, wherein said secondcomposition is a pharmaceutical composition.
 33. The combination ofclaim 29, wherein said at least a second anti-cancer agent is admixedwith said at least a first targeting agent-therapeutic agent constructto form a therapeutic cocktail.
 34. The combination of claim 29, whereinsaid at least a second anti-cancer agent is comprised within acomposition distinct from said at least a first targetingagent-therapeutic agent construct.
 35. The kit of claim 1, wherein saidkit comprises at least a first pharmaceutically acceptable liposomalformulation.